Direct selection of antigen-specific T cells, compositions obtained thereby and methods of use thereof

ABSTRACT

The invention provides a method for convenient analysis and cell separation of antigen-specific T cells based on one or more products secreted by these cells in response to antigen stimulation. The T cells are provided with a capture moiety for the product, which can then be used directly as a label in some instances, or the bound product can be further labeled via label moieties that bind specifically to the product and that are labeled with traditional labeling materials such as fluorophores, radioactive isotopes, chromophores or magnetic particles. The labeled cells are then separated using standard cell sorting techniques based on these labels. Such techniques include flow cytometry, magnetic gradient separation, centrifugation, and the like.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This is a continuation in part of U.S. Serial No. 60/085,136filed May 11, 1998

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

[0002] Not Applicable

TECHNICAL FIELD

[0003] The invention is in the field of analysis of cell populations andcell separation and the compositions obtained thereby. Moreparticularly, the invention concerns analysis and separation ofantigen-specific T cells based on primary labeling of cells with theirsecreted products through capture of these products by a specificbinding partner for the product anchored or bound to the cell surface.

BACKGROUND ART

[0004] Numerous attempts have been made to analyze populations of cellsand to separate cells based on the products which they produce. Suchapproaches to cell analysis and separation are especially useful inassessing those cells which are capable of secreting a desired product(the “product”), or which are relatively high secretors of the product.These methods include cloning in microtiter plates and analysis of theculture supernatant for product, cloning in agar and analysis by methodsfor identification of the product of the localized cells; theidentification methods include, for example, plaque assays and westernblotting. Most methods for analysis and selection of cells based uponproduct secretion involve physically isolating the cell, followed byincubation under conditions that allow product secretion, and screeningof the cell locations to detect the cell or cell clones that produce theproduct. When cells are in suspension, after the cells have secreted theproduct, the product diffuses from the cell without leaving a marker toallow identification of the cell from which it was secreted. Thus,secretor cells cannot be separated from non-secretor cells with thesetypes of systems.

[0005] In other cases, both secretor and non-secretor cells canassociate the product with the cell membrane. An example of this type ofsystem are B cell derived cell lines producing monoclonal antibodies. Ithas been reported that these types of cell lines were separated byfluorescence activated cell sorting (FACS) and other methods reliantupon the presence of antibody cell surface markers. However, proceduresthat analyze and separate cells by markers that are naturally associatedwith the cell surface can not accurately identify and/or be used in theseparation of secretor cells from non-secretor cells. In addition,systems such as these are not useful in identifying quantitativedifferences in secretor cells (i.e., low level secretors from high levelsecretors).

[0006] A method that has been used to overcome the problems associatedwith product diffusion from the cells has been to place the cell in amedium that inhibits the rate of diffusion from the cell. A typicalmethod has been to immobilize the cell in a gel-like medium (agar), andthen to screen the agar plates for product production using a systemreliant upon blotting, for example Western blots. These systems arecumbersome and expensive if large numbers of cells are to be analyzedfor properties of secretion, non-secretion, or amount of secretion.

[0007] Köhler et al. have described a negative-selection system in whichmutants of a hybridoma line secreting IgM with anti-trinitrophenyl(anti-TNP) specificity were enriched by coupling the hapten (i.e., TNP)to the cell surface and incubating the cells in the presence ofcomplement. In this way, cells secreting wild-type Ig were lysed,whereas cells secreting IgM with reduced lytic activity or not bindingto TNP preferentially survived. Köhler and Schulman (1980) Eur. J.Immunol. 10:467-476.

[0008] More recently, a system has been described for labeling andseparating cells based on secreted product. PCT/US93/10126. In thissystem, a specific binding partner for a secreted product is coupled tothe surface of cells. The product is secreted, released, and bound tothe cell by the specific binding partner. Cells are then separated basedon the degree to which they are labeled with the bound product.

[0009] Other systems allow the cells to secrete their products in thecontext of microdroplets of agarose gel which contain reagents that bindthe secretion products, and encapsulation of the cells. Such methodshave been disclosed in publications by Nir et al. (1990) Applied andEnviron. Microbiol. 56:2870-2875; and Nir et al. (1991) Applied andEnviron. Microbiol. 56:3861-3866. These methods are unsatisfactory for avariety of reasons. In the process of microencapsulation, statisticaltrapping of numbers of cells in the capsules occurs, resulting in eithera high number of empty capsules when encapsulation occurs at low cellconcentrations, or multiple cells per capsule when encapsulation occursat high cell concentrations. Secreted product is trapped in the agarosedrop by the capture antibody and detected by a second fluorochromatedantibody. This process, while allowing for the detection and isolationof cells based on secreted product, is complicated, requires specialequipment, and is not suited to all types of sorting methods. In orderto analyze and separate single cells or single cell clusters by thistechnique, large volumes must be handled to work with relatively smallnumbers of cells because of the numbers of empty capsules and because ofthe size of the microcapsules (50-100 μm). The large volume of dropletsresults in background problems using flow cytometry analysis andseparation. In addition, the capsules do not allow separation usingmagnetic beads or panning for cell separation.

[0010] Various methods have been used to couple labels to cell surfaceswhere the label such as a fluorochrome is intended for direct detection.For example, hydrophobic linkers inserted into the cell membrane tocouple fluorescent labels to cells have been described in PCT WO90/02334, published 8 Mar. 1990. Antibodies directed to HLA have alsobeen used to bind labels to cell surfaces. Such binding results in asmaller dimension than the encapsulated droplets described above andsuch cells can be conveniently used in standard separation proceduresincluding flow cytometry and magnetic separations.

[0011] ELISpot assays and methods for intracellular cytokine staininghave been used for enumeration and characterization of antigen-specificCD4⁺ and CD8⁺ T cells. Lalvani et al. (1997) J. Exp. Med. 186:859-865;and Waldrop et al. (1997) J. Clin Invest. 99:1739-1750. These methodscan be quite useful for T-cell epitope mapping or for monitoringimmunogenicity in vaccine trials, but they do not allow isolation oflive antigen-specific T cells, e.g., for clinical trials of specificadoptive immunotherapy of cancer or infections. Kern et al. (1998) Nat.Med. 4:975-978; El Ghazali et al. (1993) Curr. Opin Immunol.23:2740-2745; and Yee et al (1997) Curr. Opin. Immund. 9:702-708.Soluble multivalent complexes of peptide-loaded major histocompatibilitycomplex (MHC) molecules have been exploited recently to detect and alsoisolate antigen-specific T cells. Altman et al. (1996) Science274:94-96; Dunbar et al. (1998) Curr. Biol. 8:413-416; Ogg et al. (1998)279:2103-2106; Luxembourg et al. (1998) Nat. Biotechnol. 16:281-285;Murali-Krishna et al. (1998) Immunity 8:177-187; Gallimore et al. (1998)J. Exp. Med. 187:1383-1393; and Flynn et al. (1998) Immunity 8:683-691.These reagents are highly specific but the approach is limited to welldefined combinations of antigenic peptides and restricting HLA alleles.

[0012] The immune system comprises two types of antigen-specific cells:B cells and T cells. T cells can be characterized phenotypically by themanner in which they recognize antigen, by their cell surface markers,and by their secreted products. Unlike B cells, which recognize solubleantigen, T cells recognize antigen only when the antigen is presented tothem in the form of small fragments bound to major histocompatibilitycomplex (MHC) molecules on the surface of another cell. Any cellexpressing MHC molecules associated with antigen fragments on itssurface can be regarded as an antigen-presenting cell (APC). In mostsituations, however, more than the mere display of an MHC-bound antigenfragment on a cell surface is required to activate a T lymphocyte. Inaddition to the signal delivered via the T cell receptor (TCR) engagedby MHC molecule plus antigen, the T cell must also receiveco-stimulatory signals from the APC. Typically APCs are dendritic cells,macrophages or activated B lymphocytes.

[0013] T cells express distinctive membrane molecules. Included amongthese are the T cell antigen receptor (TCR), which appears on the cellsurface in association with CD3; and accessory molecules such as CD5,CD28 and CD45R. Subpopulations of T cells can be distinguished by thepresence of additional membrane molecules. Thus, for example, T cellsthat express CD4 recognize antigen associated with class II MHCmolecules and generally function as helper cells, while T cells thatexpress CD8 recognize antigen associated with class I MHC molecules andgenerally function as cytotoxic cells. The CD4⁺ subpopulation of T cellscan be categorized further into at least two subsets on the basis of thetypes of cytokines secreted by the cell. Thus, while both subsetssecrete IL-3 and GM-CSF, TH1 cells generally secrete IL-2, IFN-γ, andTNF-λ, whereas TH2 cells generally secrete IL-4, IL-5, IL-10, and IL-13.

[0014] Minor changes in the peptide bound to the MHC molecule can notaffect the affinity of the peptide-MHC molecule interaction, yet theycan generate partial signals that lead to a halfway responsecharacterized by proliferation and secretion of only a fraction of thecytokines produced during a full T cell response. Some modified peptidescan even block proliferation and cytokine secretion altogether andinduce a state of T cell anergy or unresponsiveness. There are thusthree different types of peptides: agonist (those that stimulate a fullresponse), partial agonist (those that stimulate a partial response) andantagonist (those that induce unresponsiveness). When a single APCpresents a mixture of an agonist and an antagonist on its surface, thenegative effect of the latter can overcome the positive effect of theformer, even if the antagonist is present in much smaller amounts thanthe agonist. Some viruses seem to use mutations in their proteins toproduce antagonist peptides capable of suppressing the activity of the Tcell clones that recognize agonist peptides derived from the originalwild-type virus.

[0015] Secretion by a T cell of a particular cytokine is generallyassociated with a particular function. For example, differences in thecytokines secreted by the TH1 and TH2 subsets of CD4⁺ T cells arebelieved to reflect different biological functions of these two subsets.The TH1 subset is responsible for classical cell-mediated functions suchas delayed-type hypersensitivity and activation of cytotoxic T cells,whereas the TH2 subset functions more effectively as a helper for B-cellactivation. The TH1 subset can be particularly suited to respond toviral infections and intracellular pathogens because it secretes IL-2and IFN-γ, which activate cytotoxic T cells. The TH2 subset can be moresuited to respond to extracellular bacteria and helminthic parasites andcan mediate allergic reactions, since IL-4 and IL-5 are known to induceIgE production and eosinophil activation, respectively. There is alsoconsiderable evidence suggesting that preferential activation of TH1cells plays a central role in the pathogenesis of a number of autoimmunediseases. Secretion of IL-10 by TH2 cells is thought to suppress, in anindirect manner, cytokine production by TH1 cells, and, accordingly, hasa general immunosuppressive effect. A shift in the TH1/TH2 balance canresult in an allergic response, for example, or, in an increasedcytotoxic T cell response.

[0016] The changes initiated by the TCR in the first few minutes tohours of activation lead to transition of the cell from the G0 to G1phase of the cell cycle. Several hours after stimulation of the T cellbegins to express IL-2 and high-affinity IL-2 receptor. IL2 geneexpression is effected by a set of transcription factors that areactivated by the converging signaling pathways triggered by the ligationof TCR, CD28 and possibly other T cell surface molecules.

[0017] The transcription factors also induce expression of the CD25gene, which encodes the α-subunit of the high-affinity IL-2 receptor.The interaction of IL-2 with the high-affinity receptor initiatessignaling pathways that cause the T cell to transit from the G1 to the Sphase of the cell cycle and progress to cell division. The signalingpathways control the expression and activity of several key proteinsnecessary for cell division. Some of these are also activated directlyby TCR- and CD28-dependent signals while others are energized only bysignals provided via the IL-2 receptor.

[0018] The stimulated T cell undergoes a sequence of phenotypic changesbeginning with its progression from the resting state to mitosis andlater to differentiation into effector and memory cells. Among theearliest (immediate) changes, observable within 15-30 minutes ofstimulation, are the expression of genes encoding transcription factorssuch as c-Fos, NF-AT, c-Myc and NF-κB, protein kinases such as Jak-3 andprotein phosphatases such as Pac-1. The subsequent early changes,occurring within several hours of stimulation, mark the beginning of theexpression of genes encoding activation antigens. These include severalcytokines (IL-2 and others), IL-2 receptor subunit α (CD25), insulinreceptor, transferrin receptor and several other surface molecules suchas CD 26, CD30, CD54, CD69 and CD70.

[0019] Activation antigens reach a maximum level of expression justbefore the first division, 24 hours after stimulation. During thisperiod the level of expression of several other molecules alreadyexpressed on resting T cells increases. At a later point, some daysafter activation commenced, late activation antigens become expressed onthe T cells. These include MHC class II molecules and several members ofthe β1 integrin family. Expression of late activation antigens marks thedifferentiation of the activated cell into effector or memory T cells.

[0020] T cells play important roles in autoimmunity, inflammation,cytotoxicity, graft rejection, allergy, delayed-type hypersensitivity,IgE-mediated hypersensitivity, and modulation of the humoral response.Disease states can result from the activation of self-reactive T cells,from the activation of T cells that provoke allergic reactions, or fromthe activation of autoreactive T cells following certain bacterial andparasitic infections, which can produce antigens that mimic humanprotein, rendering these protein “autoantigens.” These diseases include,for example, the autoimmune diseases, autoimmune disorders that occur asa secondary event to infection with certain bacteria or parasites, Tcell-mediated allergies, and certain skin diseases such as psoriasis andvasculitis. Furthermore, undesired rejection of a foreign antigen canresult in graft rejection or even infertility, and such rejection can bedue to activation of specific T lymphocyte populations. Pathologicalconditions can also arise from an inadequate T cell response to a tumoror a viral infection. In these cases, it would be desirable to increasean antigen-specific T cell response in order to reduce or eliminate thetumor or to eradicate an infection.

[0021] Autoimmune diseases have a variety of causes. For instance,autoimmune reactions can be provoked by injury or immunization withcollagen, by superantigens, by genetic factors, or errors in immuneregulation. Superantigens are polyclonal activators that can, amongother things, stimulate clones previously anergized by an encounter withan autoantigen or clones that ignored the potential autoantigens becauseof their low expression or availability. Certain autoimmune disease arecaused mainly by autoantibodies, others are T cell-mediated.Autoreactive T cells cause tissue damage in a number of autoimmunediseases including rheumatoid arthritis and multiple sclerosis.

[0022] In the treatment of autoimmune disorders, nonspecific immunesuppressive agents have been used to slow the disease; these therapiesoften cause a general immunosuppression by randomly killing orinhibiting immunocompetent cells. Attempts to treat autoimmune disordersby modulating the activity of autoreactive T cells have includedimmunization with TCR peptides, treatment with interferon-β (IFN-β) andT lymphocyte vaccination. Ebers (1994) Lancet 343:275-278; Hohlfeld(1997) Brain 120:865-916; and Hafler et al. (1992) Clin. Immunol.Immunopathol . 62:307-313.

[0023] The development of allergic sensitization, contact sensitivityand inflammation is dependent on activation and stimulation of T cellsthat exhibit proallergic functions. Allergen-specific T cells arebelieved to play an important role in the pathophysiology of atopicallergies. Elimination or suppression of allergen-specific T cells couldhelp ameliorate allergic diseases caused by such T cells.

[0024] In the initial phase of an allergic reaction, antigen (allergen)enters the body, is picked up by APCs, displayed by them in the contextof class II MHC molecules and recognized by helper T cell precursors.These are stimulated to proliferate and differentiate mainly into TH2cells, which help B lymphocytes differentiate into antibody-producingplasma cells. As in any other antibody-mediated response, the B cellsthat receive specific help from TH cells are those that recognized theallergen via their surface receptors. Some of the cytokines produced bythe TH2 cells, especially IL-4 and IL-13, stimulate the B cells toeffect an immunoglobulin isotype switch and to produce IgE antibodies.The antibodies bind to high-affinity Fc receptors on the surface of mastcells in the connective tissue and mucosa, as well as to those on thesurface of basophils in the circulation and mucosa and initiate themanifestations of allergic reaction.

[0025] Allograft rejection is caused principally by a cell-mediatedimmune response to alloantigens (primarily MHC molecules) expressed oncells of the graft. Analysis of the T lymphocyte subpopulations involvedin allograft rejection has implicated both CD4⁺ and CD8⁺ populations.TH1 cells initiate the inflammatory reaction of delayed-typehypersensitivity, leading to the recruitment of monocytes andmacrophages into the graft. Natural kill (NK) cells, presumably alertedby the absence in the graft of MHC molecules present in the recipient,can also attack the graft in the early phases of the response.Neutrophils are mainly responsible for clearing the wound or removingdamaged cells and cellular debris in the late phase of the allograftreaction.

[0026] Most immunosuppressive treatments developed have the disadvantageof being non-specific; that is, they result in generalizedimmunosuppression, which places the recipient at increased risk forinfection. Immunosuppressive agents employed to prevent organ rejectioninclude mitotic inhibitors such as azathioprine, cyclophosphamide andmethotrexate; corticosteroids; and drugs, such as cyclosporin, FK506 andrapamycin, which inhibit the transcription of the genes encoding IL-2and the high-affinity receptor for IL-2.

[0027] In the treatment of cancers, cellular immunotherapy has beenemployed as an alternative, or an adjunct to, conventional therapiessuch as chemotherapy and radiation therapy. For example, cytotoxic Tlymphocyte (CTL) responses can be directed against antigens specificallyor preferentially presented by tumor cells. Following activation with Tcell cytokines in the presence of appropriately presented tumor antigen,tumor infiltrating lymphocytes (TILs) proliferate in culture and acquirepotent anti-tumor cytolytic properties. Weidmann et al. (1994) CancerImmunol. Immunother. 39:1-14.

[0028] The introduction into a cancer patient of in vitro activatedlymphocyte populations has yielded some success. Adoptive immunotherapy,the infusion of immunologically active cells into a cancer patient inorder to effect tumor regression, has been an attractive approach tocancer therapy for several decades. Two general approaches have beenpursued. In the first, donor cells are collected that are eithernaturally reactive against the host's tumor, based on differences in theexpression of histocompatibility antigens, or made to be reactive usinga variety of “immunizing” techniques. These activated donor cells arethen transfused to a tumor-bearing host. In the second general approach,lymphocytes from a cancer patient are collected, activated ex vivoagainst the tumor and then reinfused into the patient. Triozzi (1993)Stem Cells 11:204-211; and Sussman et al. (1994) Annals Surg. Oncol.1:296.

[0029] Current methods of cancer treatment are relatively non-selective.Surgery removes the diseased tissue, radiotherapy shrinks solid tumorsand chemotherapy kills rapidly dividing cells. Systemic delivery ofchemotherapeutic agents, in particular, results in numerous sideeffects, in some cases severe enough to preclude the use of potentiallyeffective drugs.

[0030] Viral diseases are also candidates for immunotherapy. Heslop etal. (1996) Nature Med. 2:551-555. Immunological responses to viralpathogens are sometimes ineffective in eradicating or sufficientlydepleting the virus. Furthermore, the highly mutable nature of certainviruses, such as human immunodeficiency virus, allows them to evade theimmune system.

[0031] Clearly, there is a need to identify, analyze and enrichpopulations of T cells involved in the above-mentioned pathologies.Currently, several methods for analysis and for enrichment ofantigen-specific and/or cytokine-secreting T cells exist. Enrichment ofantigen-specific T cells can be achieved using selective culturingtechniques to obtain T cell lines and T cell clones. These techniquesgenerally involve culturing the T cells in vitro over a period ofseveral weeks and using rather cumbersome methods to select lines orclones exhibiting the desired phenotype, such as cytokine secretion.Other attempts to detect and enrich for antigen-specific T cells haveemployed defined multimeric MHC-antigen and MHC-peptide complexes. U.S.Pat. No. 5,635,363. For such a technique to be successful, however,MHC-antigen complexes of the correct MHC allotype are required, and theselection is limited to antigen specificity, i.e., no selection forcytokine secretion is afforded by this technique.

[0032] Intracellular cytokine staining after antigen activation,followed by FACS analysis, is the method used to obtain informationregarding the antigen specificity and kinetics of cytokine production.Waldrop et al. (1997) J. Clin. Invest. 99:1739-1750. This method isuseful for analysis only, since the cells are not viable after thisprocedure. Similarly, cytokine ELISPOT assays are useful for analysisonly. Miyahira et al. (1995) J. Immunol. Met. 181:45-54; and Lalvani etal. (1997) J. Exp. Med. 186:859-865. In these assays, secreted cytokinesare trapped in a surrounding matrix for analysis, but there is nomechanism for identifying and retrieving the cell which secreted thecytokine. The gel microdrop technology is not suited to processing largenumbers of cells such as would be necessary for treatment of theabove-mentioned indications.

[0033] It is apparent from the foregoing discussion that there is a needfor reliable techniques for analyzing and separating populations of Tcells, based on secreted product, for a number of therapeutic anddiagnostic purposes. The present invention addresses this need byproviding methods for analyzing, separating and enriching populations ofantigen-specific T cells.

DISCLOSURE OF THE INVENTION

[0034] The invention provides a method for convenient analysis and cellseparation of antigen-specific T cells based on one or more productssecreted by these cells in response to antigen stimulation. The T cellsare provided with a capture moiety specific for the product (or,“specific binding partner”), which can then be used directly as a label.The binding of the product to the capture moiety results in a “capturedproduct.” Alternatively, the cells are bound to the product via thecapture moiety and can be further labeled via label moieties that bindspecifically to the product and that are, in turn, labeled eitherdirectly or indirectly with traditional labeling materials such asfluorophores, radioactive isotopes, chromophores or magnetic particles.

[0035] The labeled cells can then be separated using standard cellsorting techniques based on these labels. Such techniques include, butare not limited to, flow cytometry, FACS, high gradient magneticgradient separation, centrifugation.

[0036] Thus, in one aspect, the invention encompasses a method tostimulate and separate antigen-specific T cells from a population ofcells according to a product secreted and released by the antigenspecific T cells in response to the stimulation. The method comprisesstimulating a mixture of cells containing T cells with antigen, andeffecting a separation of antigen-stimulated cells according to thedegree to which they are labeled with the product. Antigen stimulationis achieved by exposing the cells to at least one antigen underconditions effective to elicit antigen-specific stimulation of at leastone T cell. Labeling with the product is achieved by modifying thesurface of the cells to contain at least one capture moiety, culturingthe cells under conditions in which the product is secreted, releasedand specifically bound (“captured” or “entrapped”) to said capturemoiety; and labeling the captured product with a label moiety, where thelabeled cells are not lysed as part of the labeling procedure or as partof the separation procedure.

[0037] Another aspect of the invention is a composition of mattercontaining antigen-specific T cells capable of capturing a productsecreted and released by these cells in response to antigen stimulation,where the surface of the cells is modified to contain a capture moietyfor the product. The captured product can be separately labeled by alabel moiety.

[0038] Still another aspect of the invention is antigen-specific T cellsand progeny thereof separated by the above-described method.

[0039] Yet another aspect of the invention is a method to labelantigen-specific T cells with a product secreted and released by thecells in response to antigen stimulation, by modifying the surface ofthese cells to contain a specific binding partner for the productcoupled to the cell surface, and culturing the cells under conditionswherein the product is secreted and released.

[0040] An additional aspect of the invention is a method of analyzing apopulation of antigen-specific T cells to determine the proportion ofcells that secrete an amount of product relative to other cells in thepopulation, where the product is secreted in response to antigenstimulation. The method comprises labeling the cells by theabove-described method, further labeling the cells with a second labelthat does not label the captured product, and detecting the amount ofproduct label relative to the second cell label. Such methods areuseful, for example, in assessing the immune status of an individual.

[0041] A further aspect of the invention is methods for use of T cellpopulations enriched in antigen-specific T cells. The methods compriseadministering to an individual in need of treatment a compositioncomprising a T cell population enriched in antigen-specific T cells.Such methods are useful to treat a variety of pathological conditions,including cancer, allergies, immunodeficiencies, autoimmune disordersand viral diseases.

[0042] Yet another aspect of the invention is a kit for use inseparation of antigen-specific T cells from a mixed populationcomprising effector cells. The kit can contain a physiologicallyacceptable medium which can be of varying degrees of viscosity up to agel-like consistency, a product capture system comprising anchor andcapture moieties; a label system for detecting the captured product; andinstructions for use of the reagents, all packaged in appropriatecontainers. Optionally, the kit further comprises a magnetic labelingsystem and/or one or more biological modifiers.

[0043] Still another aspect of the invention is a kit for use in thedetection/separation of antigen-specific T cells that secrete a desiredproduct in response to antigen stimulation, the kit comprising a productcapture system comprising anchor and capture moieties; a label systemfor detecting the captured product; and instructions for use of thereagents, all packaged in appropriate containers. Optionally, the kitfurther comprises a magnetic labeling system, and/or antigen, and/or oneor more biological modifiers.

BRIEF DESCRIPTION OF THE DRAWINGS

[0044] FIGS. 1A-P are FACS plots showing analysis of cells subjected tothe separation protocol described in Example 1. A-H show analysis ofcontrol cells cultured with no peptide; I-P show analysis ofpeptide-stimulated cells. A, C, I, and K show scatter properties of thestarting cell population (A and I) and the enriched cell population (Cand K). B, D, J and L show profiles of PI versus PE staining of thestarting cell population (B and J) and the enriched cell population (Dand L). Plots E-H and M-P show FITC-labeled anti-CD8 versus PE-labeledanti-IFN-γ staining of the starting cell population (E and M), the firstnegative population (F and N), the second negative population (G and O)and the enriched cell population (H and P).

[0045] FIGS. 2A-N are FACS plots showing analysis of cells subjected tothe separation protocol described in Example 2. A-G show analysis ofcontrol cells cultured with no peptide; N-R show analysis ofpeptide-stimulated cells. A-D and H-K show FITC-labeled anti-CD8 versusPE-labeled anti-IFN-γ staining of the starting cell population (A andJ), the first negative population (B and I), the second negativepopulation (C and J) and the enriched cell population (D and K). F and Mshow staining for Vβ17TCR of the enriched cell population.

[0046]FIG. 3 is a series of dot plots showing IFN-γ-secretion-basedenrichment and detection of live antigen-specific CD4⁺ and CD8⁺ T cells.Dot plots show CD8-Cy5 vs. anti IFN-γ-PE (A-D) or CD4-Cy5 vs. antiIFN-γ-PE (E-L) staining of PBMC from healthy adult donors stimulatedwith (A,B) or without (C,D) the HLA-A0201-restricted FLU 58-66 peptide,a purified influenza A virus preparation (with (E,F) without (G,H)) andrTT.C (with (I,J) without (K,L)) before (A,C,E,G,I,K) and after(B,D,F,H,J,L) magnetic enrichment of IFN-γ-secreting cells. Livelymphocytes were gated according to light-scatter properties andpropidium iodide exclusion.

[0047]FIG. 4 is a series of dot plots showing a phenotypic analysis ofenriched Flu 58-66 peptide-specific CD8⁺ T cells. EnrichedIFN-γ-secreting CD8⁺ T cells from FLU 58-66 peptide-stimulated PBMC(A,B,E,F) and, for control, from non-stimulated PBMC (C,D,G,H) werestained with anti IFN-γ-PE and counterstained with FITC-conjugatedantibodies against CD27, CD28, CD57 and the TCR Vβ17 chain.Light-scatter properties, propidium iodide and CD8-Cy5 staining wereused for gating of live CD8⁺ T cells.

[0048]FIG. 5 is a graph depicting cytolytic activity of enriched andexpanded Flu 58-66 peptide-specific T cells. IFN-γ-secreting CD8⁺ Tcells from FLU 58-66 peptide-stimulated PBMC were expanded for 18 daysin tissue culture in the presence of IL-2 and then assayed for CTLactivity assay. The diagram shows the percentage of lysed HLA-A2.1+T2cells pulsed with either Flu 58-66 peptide or the negative controlpeptide Melan A/MART 1 27-35.

[0049]FIG. 6 is a series of dot plots depicting the isolation anddetection of TT-specific IL-4-secreting CD4+T cells. Dot plots showCD4-Cy5 vs. anti IL-4-PE staining of PBMC from healthy adult donorsstimulated with (A,C) or without (B,D) magnetic enrichment ofIL-4-secreting cells. Live lymphocytes were gated according tolight-scatter properties and propidium iodide exclusion.

MODES FOR CARRYING OUT THE INVENTION

[0050] The present invention provides methods for detecting, analyzingand separating antigen-stimulated T cells on the basis of secretedproduct, where the product is secreted as a result of antigenstimulation. The methods are based on capture and relocation to the cellsurface of the secreted product.

[0051] The captured product permits the cell to be detected, analyzedand, if desired, sorted, according to the presence, absence or amount ofthe product present. The means of capture comprises a product-specificbinding partner (“capture moiety”) anchored to the cell surface by ameans suitable for the cell to be sorted.

[0052] The approach presented here combines, inter alia, the followingadvantages: (a) it permits rapid isolation, enumeration, phenotyping andexpansion of live antigen-specific T lymphocytes without the need ofcyclical activation of T cells with antigen and APCs; (b) it isgenerally applicable for isolation of T cells reactive to APCs that havebeen pulsed with synthetic peptides, native proteins, cell extracts,nonviable pathogens, transduced with retroviral vectors, infected withrecombinant viral vectors, transfected with RNA or DNA, etc.; (c) it canbe used for the isolation of both CD4⁺ antigen-specific Th cells andCD8⁺ antigen-specific CTLs; and (d) it enables selective isolation ofantigen specific T cells with particular cytokinemediated effectorfunctions, e.g., of antigen-specific Th1-, Th2-, or Th3-likelymphocytes.

[0053] The practice of the present invention will employ, unlessotherwise indicated, conventional techniques of molecular biology(including recombinant techniques), microbiology, cell biology,biochemistry and immunology, which are within the skill of the art. Suchtechniques are explained fully in the literature, such as, “MolecularCloning: A Laboratory Manual”, second edition (Sambrook et al., 1989);“Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal CellCulture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (AcademicPress, Inc.); “Handbook of Experimental Immunology” (D. M. Weir & C. C.Blackwell, eds.); “Gene Transfer Vectors for Mammalian Cells” (J. M.Miller & M. P. Calos, eds., 1987); “Current Protocols in MolecularBiology” (F. M. Ausubel et al., eds., 1987, and periodic updates); “PCR:The Polymerase Chain Reaction”, (Mullis et al., eds., 1994); and“Current Protocols in Immunology” (J. E. Coligan et al., eds., 1991).

[0054] Cell sorting and cell analysis methods are known in the art andare described in, for example, The Handbook of Experimental Immunology,Volumes 1 to 4, (D. N. Weir, editor) and Flow Cytometry and Cell Sorting(A. Radbruch, editor, Springer Verlag, 1992).

[0055] As used herein, a “specific binding partner” or “capture moiety”intends a member of a pair of molecules (a “specific binding pair”) thatinteract by means of specific non-covalent interactions that depend onthe three-dimensional structures of the molecules involved. A “labelmoiety” is a detectable, either directly or indirectly. When the capturemoiety is an antibody, it can be referred to as the “capture antibody”or “catch antibody.” The capture moieties are those which attach both tothe cell, either directly or indirectly, and the product. The labelmoieties are those which attach to the product and can be directly orindirectly labeled.

[0056] As used herein, the term “antibody” is intended to includepolyclonal and monoclonal antibodies, chimeric antibodies, haptens andantibody fragments, and molecules which are antibody equivalents in thatthey specifically bind to an epitope on the product antigen. The term“antibody” includes polyclonal and monoclonal antibodies of any isotype(IgA, IgG, IgE, IgD, IgM), or an antigen-binding portion thereof,including, but not limited to, F(ab) and Fv fragments such as sc Fv,single chain antibodies, chimeric antibodies, humanized antibodies, anda Fab expression library. Antibodies can also be immobilized forinstance on a polymer or a particle.

[0057] “Bispecific antibody” and “bispecific antibodies,” also known asbifunctional antibodies, intends antibodies that recognize two differentantigens by virtue of possessing at least one first antigen combiningsite specific for a first antigen or hapten, and at least one secondantigen combining site specific for a second antigen or hapten. Suchantibodies can be produced by recombinant DNA methods or include, butare not limited to, antibodies chemically by methods known in the art.Chemically created bispecific antibodies that have been reduced andreformed so as to retain their bivalent characteristics and antibodiesthat have been chemically coupled so that they have at least two antigenrecognition sites for each antigen. Bispecific antibodies include allantibodies or conjugates of antibodies, or polymeric forms of antibodieswhich are capable of recognizing two different antigens. The labelmoiety can be a fluorochromated antiproduct antibody, which can include,but is not limited to, magnetic bead conjugated, colloidal beadconjugated, FITC, Phycoerythrin, PerCP, AMCA, fluorescent particle orliposome conjugated antibodies. Alternatively the label moiety can beany suitable label including but not limited to those described herein.Bispecific antibodies include antibodies that have been reduced andreformed so as to retain their bivalent characteristics and toantibodies that have been chemically coupled so that they can haveseveral antigen recognition sites for each antigen.

[0058] As used herein the term “effector cell population” intends a cellpopulation which comprises at least one T cell. An effector cellpopulation can be obtained from a starting cell population from whichantigen-specific T cells are enriched.

[0059] The terms “cell,” and “cells,” and “cell population,” usedinterchangeably, intend one or more mammalian cells. The term includesprogeny of a cell or cell population. Those skilled in the art willrecognize that “cells” include progeny of a single cell, and the progenycan not necessarily be completely identical (in morphology or of totalDNA complement) to the original parent cell due to natural, accidental,or deliberate mutation and/or change.

[0060] The terms “T lymphocyte,” “T cell,” “T cells,” and “T cellpopulation,” used interchangeably, intends a cell or cells which displayon their surface one or more antigens characteristic of T cells, suchas, for example, CD2 and CD3. The term includes progeny of a T cell or Tcell population. A “T lymphocyte” or “T cell” is a cell which expressesCD3 on its cell surface and a T cell antigen receptor (TCR) capable ofrecognizing antigen when displayed on the surface of autologous cells,or any antigen-presenting matrix, together with one or more MHCmolecules or, one or more non-classical MHC molecules. The term “Tcells” as used herein denotes any T cells known in the art, forinstance, lymphocytes that are phenotypically CD3⁺, i.e., express CD3 onthe cell surface, typically detected using an anti-CD3 monoclonalantibody in combination with a suitable labeling technique. The T cellsenriched by the methods of this invention are generally CD3⁺. The Tcells enriched by the methods of this invention are also generally,although not necessarily, positive for CD4, CD8, or both.

[0061] The term “substantially enriched” as used herein, indicates thata cell population is at least about 50-fold, more preferably at leastabout 500-fold, and even more preferably at least about 5000-fold ormore enriched from an original mixed cell population comprising thedesired cell population.

[0062] The term “antigen-presenting matrix,” as used herein, intends amolecule or molecules which can present antigen in such a way that theantigen can be bound by a T cell antigen receptor on the surface of a Tcell. An antigen-presenting matrix can be on the surface of anantigen-presenting cell (APC), on a vesicle preparation of an APC, orcan be in the form of a synthetic matrix on a bead or a plate. The term“antigen presenting cell”, as used herein, intends any cell whichpresents on its surface an antigen in association with a MHC or portionthereof, or, one or more nonclassical MHC molecules, or a portionthereof.

[0063] The term “autogeneic,” “autologous,” or, “self,” as used herein,indicates the origin of a cell. Thus, a cell is autogeneic if the cellwas derived from an individual (the “donor”) or a genetically identicalindividual and is to be readministered to the individual. An autogeneiccell can also be a progeny of an autogeneic cell. The term alsoindicates that cells of different cell types are derived from the samedonor or genetically identical donors. Thus, an effector cell and anantigen presenting cell are said to be autogeneic if they were derivedfrom the same donor or from an individual genetically identical to thedonor, or if they are progeny of cells derived from the same donor orfrom an individual genetically identical to the donor.

[0064] Similarly, the term “allogeneic,” or “non-self,” as used herein,indicates the origin of a cell. Thus, a cell and progeny thereof isallogeneic if the cell was derived from an individual not geneticallyidentical to the recipient to whom it is administered; in particular,the term relates to non-identity in expressed MHC molecules. The termalso indicates that cells of different cell types are derived fromgenetically non-identical donors, or if they are progeny of cellsderived from genetically non-identical donors. For example, an APC issaid to be allogeneic to an effector cell if they are derived fromgenetically non-identical donors.

[0065] A “disease or condition related to a population ofantigen-specific T cells” is one which can be related to a population ofantigen-specific T cells or lack of adequate numbers thereof, andincludes, for example, autoimmune diseases in which antigen-specific Tcells are primarily responsible for the pathogenesis of the disease;cancers, in which cancerous cell growth is not adequately controlled bytumor-specific cytotoxic T cells; viral diseases, in whichvirus-infected cells are not lysed by cytotoxic T cells; allergies, inwhich T cells specific for allergens mediate undesired effects;immunodeficiencies, in which inadequate numbers of T cells are presentin an individual due to either infection (such as HIV) or congenitally(such as DiGeorge syndrome). It is also one in which antigen-specific Tcells modulate or regulate the activity of another cell or cellpopulation which is primarily responsible for a disease state; it isalso one in which the- presence of a population of antigen-specific Tcells is not the primary cause of the disease, but which plays a keyrole in the pathogenesis of the disease; it is also one in which apopulation of antigen-specific T cells mediates an undesired rejectionof a foreign antigen.

[0066] An “individual” is a vertebrate, preferably a mammal, morepreferably a human. Mammals include, but are not limited to, humans,farm animals, sport animals, and pets.

[0067] An “effective amount” is an amount sufficient to effectbeneficial or desired clinical results. An effective amount can beadministered in one or more administrations. For purposes of thisinvention, an effective amount of antigen-specific T cells is an amountthat is sufficient to diagnose, palliate, ameliorate, stabilize,reverse, slow or delay the progression of the disease state.

[0068] As used herein, “treatment” is an approach for obtainingbeneficial or desired clinical results. For purposes of this invention,beneficial or desired clinical results include, but are not limited to,alleviation of symptoms, diminishment of extent of disease, stabilized(i.e., not worsening) state of disease, preventing spread (i.e.,metastasis) of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, and remission (whetherpartial or total), whether detectable or undetectable. “Treatment” canalso mean prolonging survival as compared to expected survival if notreceiving treatment.

[0069] “Palliating” a disease means that the extent and/or undesirableclinical manifestations of a disease state are lessened and/or timecourse of the progression is slowed or lengthened, as compared to notadministering enriched T cell populations of the present invention.

[0070] The present invention provides methods for obtaining a cellpopulation enriched in antigen-specific T cells which secrete a product,where the product is secreted as a result of antigen stimulation. Themethods generally involve obtaining a mixed population of cellscomprising T cells; exposing the cell population to at least one antigenunder conditions effective to elicit antigen-specific stimulation of atleast one T cell; modifying the surface of said mixed population tocontain attached thereto a specific binding partner for the product;allowing expression of at least one product by the stimulated T cells,wherein the product is secreted in response to the stimulation; allowingbinding of the product to a capture moiety coupled to the surface of thecell to form a cell bound capture moiety-product complex, therebylabeling the cells; and separating the stimulated T cells according tothe degree to which they are labeled with said product.

[0071] Of course, modification of the cell surface with a specificbinding partner can be carried out before, during, or after antigenstimulation.

[0072] Antigen Presenting Matrices and Effector Cell Populations

[0073] The present invention provides methods for obtaining a cellpopulation enriched in antigen-specific T cells which secrete a productin response to antigen stimulation. The methods comprise obtaining amixed population of cells (i.e., an “effector cell population”), andexposing the cell population to at least one antigen. The mixed cellpopulation can be obtained by any method known in the art and ispreferably enriched for T cells. Exposure to antigen can be achievedusing antigen-presenting matrices, which can be on the surface ofantigen-presenting cells (APC's). Antigen-presenting matrices andeffector cells can be obtained from a variety of sources. The mixedpopulation of cells can be stimulated by antigen in vitro or in vivo, ormodified in any of a variety of ways, for example, chemically orgenetically modified.

[0074] Antigen Presenting Matrices

[0075] The T cell populations which are subjected to the methods of thepresent invention are exposed to at least one antigen under conditionseffective to elicit antigen-specific stimulation. A T cell which isstimulated by the at least one antigen is said to be antigen specific,i.e., it displays on its cell surface an antigen receptor whichspecifically recognizes and binds to an antigen in association with amolecule capable of presenting antigen, such as a classical ornon-classical MHC molecule or a portion thereof, on anantigen-presenting matrix, for example, a synthetic antigen-presentingmatrix or one that is present on the surface of an APC.

[0076] The antigen-presenting molecule can be an MHC molecule, which canbe class I or class II or, a non-classical MHC molecule such as CD1; anMHC epitope; a fusion protein comprising an MHC epitope; or a syntheticMHC epitope. The nature of the antigen-presenting molecule is notcritical, so long as it is capable of presenting antigen to an effectorcell. Methods of preparing MHC epitopes are known in the art.

[0077] Antigen-presenting matrices include those on the surface of anAPC as well as synthetic antigen-presenting matrices. APCs suitable foruse in the present invention are capable of presenting exogenous peptideor protein or endogenous antigen to T cells in association with anantigen-presenting molecule, such as an MHC molecule. APCs include, butare not limited to, macrophages, dendritic cells, CD40-activated Bcells, antigen-specific B cells, tumor cells, virus-infected cells andgenetically modified cells.

[0078] APCs can be obtained from a variety of sources, including but notlimited to, peripheral blood mononuclear cells (PBMC), whole blood orfractions thereof containing mixed populations, spleen cells, bonemarrow cells, tumor infiltrating lymphocytes, cells obtained byleukapheresis, lymph nodes, e.g., lymph nodes draining from a tumor.Suitable donors include an immunized donor, a non-immunized (naive)donor, treated or untreated donors. A “treated” donor is one that hasbeen exposed to one or more biological modifiers. An “untreated” donorhas not been exposed to one or more biological modifiers. APC's can alsobe treated in vitro with one or more biological modifiers.

[0079] The APCs are generally alive but can also be irradiated,mitomycin C treated, attenuated, or chemically fixed. Further, the APCsneed not be whole cells. Instead, vesicle preparations of APCs can beused.

[0080] APCs can be genetically modified, i.e., transfected with arecombinant polynucleotide construct such that they express apolypeptide or an RNA molecule which they would not normally express orwould normally express at lower levels. Examples of polynucleotidesinclude, but are not limited to, those which encode an MHC molecule; aco-stimulatory molecule such as B7; or an antigen. For example,expression of a polynucleotide encoding an MHC molecule undertranscriptional control of a strong promoter such as the CMV promoter,can result in high level expression of the MHC molecule on the cellsurface, thus increasing the density of antigen presentation.Alternatively, an APC can be transfected with a polynucleotide constructcomprising a polynucleotide encoding an antigen under transcriptionalcontrol of a strong promoter such as the CMV promoter such that theantigen is expressed on the cell surface together with an MHC molecule.

[0081] The nucleotide sequence encoding a polypeptide is operably linkedto control sequences for transcription and translation. A controlsequence is “operably linked” to a coding sequence if the controlsequence regulates transcription or translation. Any method in the artcan be used for the transformation, or insertion, of an exogenouspolynucleotide into an APC, for example, lipofection, transduction,infection or electroporation, using either purified DNA, viral vectors,or DNA or RNA viruses. The exogenous polynucleotide can be maintained asa non-integrated vector, for example, a plasmid, or, can be integratedinto the host cell genome.

[0082] Cells which do not normally function in vivo in mammals as APCscan be modified to function as APCs. A wide variety of cells canfunction as APCs when appropriately modified. Examples of such cells areinsect cells, for example Drosophila or Spodoptera; foster cells, suchas the human cell line T2, which bears a mutation in its antigenpresenting pathway that restricts the association of endogenous peptideswith cell surface MHC class I molecules. Zweerink et al. (1993) J.Immunol. 150:1763-1771. For example, expression vectors which direct thesynthesis of one or more antigen-presenting polypeptides, such as MHCmolecules, and, optionally, accessory molecules such as B7, can beintroduced into these cells to effect the expression on the surface ofthese cells antigen presentation molecules and, optionally, accessorymolecules or functional portions thereof. Alternatively,antigen-presenting polypeptides and accessory molecules which can insertthemselves into the cell membrane can be used. For example,glycosyl-phosphotidylinositol (GPI)-modified polypeptides can insertthemselves into the membranes of cells. Medof et al. J. Exp. Med.160:1558-1578; and Huang et al. Immunity 1:607-613. Accessory moleculesinclude, but are not limited to, co-stimulatory antibodies such asantibodies specific for CD28, CD80, or CD86; costimulatory molecules,including, but not limited to, B7.1 and B7.2; adhesion molecules such asICAM-1 and LFA-3; and survival molecules such as Fas ligand and CD70.See, for example, PCT Publication No. WO 97/46256.

[0083] Alternatively, a synthetic antigen-presenting matrix can be usedto present antigen to effector cells. A synthetic matrix can include anantigen presenting molecule, preferably an MHC Class I or MHC Class IImolecule, immobilized on a solid support, for example, beads or plates.Accessory molecules can be present, which can be co-immobilized orsoluble, the molecules including, but not limited to, co-stimulatoryantibodies such as antibodies specific for CD28, CD80, or CD86;costimulatory molecules, including, but not limited to, B7.1 and B7.2;adhesion molecules such as ICAM-1 and LFA-3; and survival molecules suchas Fas ligand and CD70. Portions of accessory molecules can also beused, as long as their function is maintained. Solid supports includemetals or plastics, porous materials, microbeads, microtiter plates, redblood cells, and liposomes. See, for example, PCT Publication No. WO97/46256; and WO 97/35035.

[0084] Methods for determining whether an antigen-presenting matrix,whether it is on a cell surface or on a synthetic support, is capable ofpresenting antigen to an effector cell, are known in the art andinclude, for example, ³H-thymidine uptake by effector cells, cytokineproduction by effector cells, and cytolytic ⁵¹Cr-release assays.

[0085] Effector Cell Populations

[0086] Antigen-specific T cells can be isolated from an effector cellpopulation, i.e., a population of hematopoietic cells, preferablyenriched for T cells. The effector cell population is a startingpopulation from which antigen-specific T cells are isolated.

[0087] An effector cell population suitable for use in the presentinvention can be autogeneic or allogeneic, preferably autogeneic. Wheneffector cells are allogeneic, preferably the cells are depleted ofalloreactive cells before use. This can be accomplished by any knownmeans, including, for example, mixing the allogeneic effector cells anda recipient cell population and incubating them for a suitable time,then depleting CD69⁺ cells, or inactivating alloreactive cells, orinducing anergy in the alloreactive cell population.

[0088] The effector cell population can comprise unseparated cells,i.e., a mixed population, for example, a PBMC population, whole blood,and the like. The effector cell population can be manipulated bypositive selection based on expression of cell surface markers, negativeselection based on expression of cell surface markers, stimulation withone or more antigens in vitro or in vivo, treatment with one or morebiological modifiers in vitro or in vivo, subtractive stimulation withone or more antigens or biological modifiers, or a combination of any orall of these.

[0089] Effector cells can be obtained from a variety of sources,including but not limited to, PBMC, whole blood or fractions thereofcontaining mixed populations, spleen cells, bone marrow cells, tumorinfiltrating lymphocytes, cells obtained by leukapheresis, biopsytissue, lymph nodes, e.g., lymph nodes draining from a tumor. Suitabledonors include an immunized donor, a non-immunized (naive) donor,treated or untreated donors. A “treated” donor is one that has beenexposed to one or more biological modifiers. An “untreated” donor hasnot been exposed to one or more biological modifiers.

[0090] Methods of extracting and culturing effector cells are wellknown. For example, effector cells can be obtained by leukapheresis,mechanical apheresis using a continuous flow cell separator. Forexample, lymphocytes and monocytes can be isolated from the buffy coatby any known method, including, but not limited to, separation overFicoll-Hypaque™ gradient, separation over a Percoll gradient, orelutriation. The concentration of Ficoll-Hypaque™ can be adjusted toobtain the desired population, for example, a population enriched in Tcells. Other methods based on cell-specific affinity columns are knownand can be used. These include, for example, fluorescence-activated cellsorting (FACS), cell adhesion, magnetic bead separation, and the like.Affinity-based methods can utilize antibodies, or portions thereof,which are specific for cell-surface markers and which are available froma variety of commercial sources, including, the American Type CultureCollection (Rockville, Md.). Affinity-based methods can alternativelyutilize ligands or ligand analogs, of cell surface receptors.

[0091] The effector cell population can be subjected to one or moreseparation protocols based on the expression of cell surface markers.For example, the cells can be subjected to positive selection on thebasis of expression of one or more cell surface polypeptides, including,but not limited to, “cluster of differentiation” cell surface markerssuch as CD2, CD3, CD4, CD8, TCR, CD45, CD45RO, CD45RA, CD11b, CD26,CD27, CD28, CD29, CD30, CD31, CD40L; other markers associated withlymphocyte activation, such as the lymphocyte activation gene 3 product(LAG3), signaling lymphocyte activation molecule (SLAM), T1/ST2;chemokine receptors such as CCR3, CCR4, CXCR3, CCR5, homing receptorssuch as CD62L, CD44, CLA, CD146, α4β7, αEβ7; activation markers such asCD25, CD69 and OX40; and lipoglycans presented by CD1. The effector cellpopulation can be subjected to negative selection for depletion of non-Tcells and/or particular T cell subsets. Negative selection can beperformed on the basis of cell surface expression of a variety ofmolecules, including, but not limited to, B cell markers such as CD19,and CD20; monocyte marker CD14; the NK cell marker CD56.

[0092] The effector cell population can be manipulated by exposure, invivo or in vitro, to one or more antigens. Antigens include, but are notlimited to, peptides; proteins; glycoproteins; lipids; glycolipids;cells; cell extracts; tissue extracts; whole microorganisms such asprotozoans, bacteria, and viruses. Antigens can be unmodified, i.e.,used in their native state. Alternatively, an antigen can be modified byany known means, including, but not limited to, heating, for example todenature a protein or to inactivate a pathogen; chemical modification todenature a protein, or to cross-link two antigen molecules;glycosylation; chemical modification with moieties including, but notlimited to polyethylene glycol; and enzymatic digestion. If more thanone antigen is used, the exposure can be simultaneous or sequential.

[0093] The effector cells can be cultured in the presence of at leastone antigen associated with a condition to be treated. The antigen canbe a single antigen with multiple antigenic determinants or can be amixture of antigens. The antigen can be an autoantigen or a foreignantigen, depending on the condition to be treated. Autoantigens includeantigens associated with autoimmune diseases and those associated withcancer cells. The antigen can be a protein, cells, a tissue or a targetorgan. If the antigen is an autoantigen, the autoantigen can be part ofan organ, for example the brain or the thyroid gland and need not bepurified therefrom. Purified autoantigens or mixtures of purifiedautoantigens can also be used.

[0094] Co-culturing of peripheral blood leukocytes (PBL) or tumorinfiltrating lymphocytes (TIL) with autologous tumor cells is generallyaccompanied by cytokine stimulation. Sporn et al.(1993) Cancer Immunol.Immunother. 37:175-180; and Peyret et al. (1991) Chirurgie 117:700-709.

[0095] An effector cell population can be manipulated by exposure, invivo or in vitro, to one or more biological modifiers. Suitablebiological modifiers include, but are not limited to, cytokines such asIL-2, IL-4, IL-10, TNF-α, IL-12, IFN-γ; non-specific modifiers such asphytohemagglutinin (PHA), phorbol esters such as phorbol myristateacetate (PMA), concanavalin-A, and ionomycin; antibodies specific forcell surface markers, such as anti-CD2, anti-CD3, anti-IL-2 receptor,anti-CD28; chemokines, including, for example, lymphotactin. Thebiological modifiers can be native factors obtained from naturalsources, factors produced by recombinant DNA technology, chemicallysynthesized polypeptides or other molecules, or any derivative thereofhaving the functional activity of the native factor. If more than onebiological modifier is used, the exposure can be simultaneous orsequential.

[0096] The present invention provides compositions comprising T cellsenriched in antigen-specific cells, enriched according to the methods ofthe invention. By “enriched” is meant that a cell population is at leastabout 50-fold, more preferably at least about 500-fold, and even morepreferably at least about 5000-fold or more enriched from an originalmixed cell population comprising the desired cell population. Theproportion of the enriched cell population which comprises the desiredantigen-specific cells can vary substantially, from less than 10% up to100% antigen-specific cells. The percentage which are antigen-specificcan be readily determined, for example, by a ³H-thymidine uptake assayin which the T cell population is challenged by an antigen-presentingmatrix presenting the desired antigen(s).

[0097] Cell Labeling

[0098] The methods herein are based on labeling the cells with a productsecreted by the cells, where the product is secreted in response toantigen stimulation. To achieve labeling, the cell surface of a cellpopulation is modified such that a moiety that binds specifically to aproduct, the “specific binding partner” is attached to the cell surfaceeither directly or through an anchoring means (an “anchor moiety”),optionally through a linker to form a capture moiety. The cellpopulation can contain numerous types of cells and generally made up ofa mixed population. Preferably the cell population is hematopoietic,more preferably the cell population is effector cells, most preferably,the cell population is T cells or a subset thereof. Subsets can beisolated by virtue of cell surface markers, for instance, CD45 forlymphocytes, CD8 for cytotoxic cells, etc.

[0099] Products secreted in response to antigen stimulation are known inthe art and include, but are not limited to, cytokines, such as IL-2,IL-4, IL-10, TNF-α, TGF-β and IFN-γ.

[0100] Specific binding partners include any moiety for which there is arelatively high affinity and specificity between product and bindingpartner, and in which the dissociation of the product:partner complex isrelatively slow so that the product:partner complex is detected duringthe cell separation technique. Specific binding partners include, butare not limited to, substrates or substrate analogs to which a productwill bind, peptides, polysaccharides, steroids, biotin, digitoxin,digitonin and derivatives thereof. In a preferred embodiment thespecific binding partner is an antibody or antigen-binding fragment orderivative thereof. The term “antigen-binding fragment” includes anypeptide that binds specifically to the product. Typically, thesefragments include such immunoglobulin fragments as Fab, F(ab′)₂, Fab′,scFv (both monomer and polymeric forms) and isolated H and L chains. Anantigen-binding fragment retains the specificity of the intactimmunoglobulin, although avidity and/or affinity can be altered.

[0101] In the practice of the invention the capture moiety can beattached to a cell membrane (or cell wall) by a variety of methods.Suitable methods include, but are not limited to, direct chemicalcoupling to amino groups of the protein components, coupling to thiols(formed after reduction of disulfide bridges) of the protein components,indirect coupling through antibodies (including pairs of antibodies) orlectins, anchoring in the lipid bilayer by means of a hydrophobicanchor, and binding to the negatively charged cell surface bypolycations.

[0102] In other embodiments, the capture moiety is introduced using twoor more steps, e.g., by labeling the cells with at least one anchormoiety which allows the coupling of the capture moiety to the anchormoiety either directly, for instance by a biotin/avidin complex orindirectly, through a suitable linking moiety or moieties.

[0103] Suitable anchor moieties include lipophilic molecules such asfatty acids. Alternatively, antibodies or other specific binding agentsto cell surface markers such as the MHC antigens or glycoproteins, canalso be used.

[0104] The “capture moiety” can be coupled to the anchor moiety througha linking agent, and can also include a linker which multiplies thenumber of capture moieties available and thus the potential for captureof product, such as branched polymers, including, for example, modifieddextran molecules, polyethylene glycol, polypropylene glycol, polyvinylalcohol, and polyvinylpyrrolidone.

[0105] Methods for direct chemical coupling of antibodies to the cellsurface are known in the art, and include, for example, coupling usingglutaraldehyde or maleimide activated antibodies. Methods for chemicalcoupling using multiple step procedures include, but are not limited to,biotinylation, coupling of trinitrophenol (TNP) or digoxigenin using forexample succinimide esters of these compounds. Biotinylation can beaccomplished by, for example, the use ofD-biotinyl-N-hydroxysuccinimide. Succinimide groups react effectivelywith amino groups at pH values above 7, and preferentially between aboutpH 8.0 and about pH 8.5. Biotinylation can be accomplished by, forexample, treating the cells with dithiothreitol followed by the additionof biotin maleimide.

[0106] Coupling to the cells can also be accomplished using antibodiesagainst cell surface antigens (“markers”). Antibodies directed tosurface antigens generally require in the range of 0.1 to 1 μg ofantibody per 10⁷ cells. However, this requirement will vary widely inresponse to the affinity of the antibody to the product and will need tobe determined empirically. Such a determination is well within the skillof one in the art. Thus, the appropriate amount of antibody must bedetermined empirically and is within the skill of one in the art. Thisallows coupling to specific cells on cell type specific markerexpression. For instance, classes of cells such as T cells or subsetsthereof can be specifically labeled. As a capture moiety, a bispecificantibody can be used which has an antigen recognition site for the cellor an anchor moiety placed thereon, and the product.

[0107] A capture moiety, particularly capture antibodies should beselected based on the amount of secreted product. For example, for cellswhich secrete only a few molecules, a high affinity antibody will catchmost of the secreted molecules. Alternatively, in the case where thecell secretes many molecules during the incubation time, a loweraffinity antibody can be preferred to prevent too early saturation ofthe catching matrix. Determination of suitable affinities for the levelof proteins secreted are determined empirically and are within the skillof one in the art.

[0108] Cells carrying large amounts of N-acetylneuraminic acid on theirsurface as a constituent of their lipopolysaccharides bear a negativecharge at physiological pH values. Coupling of capture moieties can bevia charge interactions. For example, moieties bearing polycations bindto negatively charged cells. Polycations are known in the art andinclude, for example, polylysine and chitosan. Chitosan is a polymerconsisting of D-glucosamine groups linked together by α-(1-4) glucosidebonds.

[0109] Another method of coupling binding partners (which can compriseone or more capture moieties) to the cells is via coupling to the cellsurface polysaccharides. Substances which bind to polysaccharides areknown in the art, and include, for example, lectins, includingconcanavalin A, solanum tuberosum, aleuria aurantia, datura stramonium,galanthus nivalis, helix pomatia, lens culinaris and other known lectinssupplied by, a number of companies, including for example, SigmaChemical Company and Aldrich Chemical Company.

[0110] In some embodiments of the invention, the product binding partneris coupled to the cell by hydrophobic anchoring to the cell membrane.Suitable hydrophobic groups that will interact with the lipid bilayer ofthe membrane are known in the art, and include, but are not limited to,fatty acids and non-ionic detergents (including, e.g., Tween-80). Adrawback to attachment of the capture moiety to the cell via theinsertion of a hydrophobic anchor is that the rate of integration of thehydrophobic moiety into the cell is low. Thus, high concentrations ofthe moiety with the hydrophobic anchor often are required. This lattersituation is often uneconomical when the capture moiety is a relativelylimited or expensive substance, for example, an antibody.

[0111] The low yield of hydrophobic molecules that embed themselves inthe membrane is relevant only when these molecules are available inrelatively limited quantities. This problem can be overcome by using abridging system that includes an anchoring partner and a partner thatcontains the capture moiety, wherein one of the partners is of higheravailability, and wherein the two parts of the bridging system have ahigh degree of specificity and affinity for each other. For example, inone embodiment avidin or streptavidin is attached to the cell surfacevia a hydrophobic anchor, while the partner with the product capturemoiety are biotinylated anti-product antibodies. In another embodiment,the cell surface is labeled with digoxigenin followed by conjugates ofanti-digoxigenin antibody fragments and anti-product antibodies. Thisapproach can be used with other pairs of molecules able to form a link,including, for example, hapten with antihapten antibodies, NTA withpolyhistidine residues, or lectins with polysaccharides. A preferredembodiment is one which allows “amplification” of the system byincreasing the number of capture moieties per anchor moiety.

[0112] In one illustrative embodiment, a branched dextran is bound topalmitic acid, thus providing a multiplicity of available binding sites.The dextran is in turn coupled to biotin and treated withavidin-conjugated antibody specific for the product.

[0113] It is of course contemplated within the embodiments of theinvention that bridging systems can be used between the anchor moietyand the capture moiety when the anchor moiety is coupled in any fashionto the cell surface. Thus, an avidin (or streptavidin) biotin linkermoiety can link an antibody anchor moiety with a capture moiety.Bispecific antibody systems can also act as linker moieties.

[0114] In order to analyze and, if desired, to select cells that havethe capability of secreting the product, cells modified as above tocontain the capture moiety are incubated under conditions that allow theproduction and secretion of the product in a sufficient amount to allowbinding to and detection of the cells that contain the captured product.These conditions are known to those of skill in the art and include,inter alia, appropriate temperature, pH, and concentrations of salts,growth factors and substrates in the incubation medium, as well as theappropriate concentrations of gas in the gaseous phase. When it isdesirable to distinguish between high and low producer cells, the timeof incubation is such that product secretion by the cells is still in alinear phase. The appropriate conditions can be determined empiricallyand such a determination is within the skill of one in the art.

[0115] Additionally, cell secretion can be modified, that is,upregulated, induced, or reduced using a biological modifier. Thebiological modifiers can be added at any time but are preferably addedto the incubation medium. Alternatively, the cells can be pretreatedwith these agents or cells prior to the incubation step. Suitablebiological modifiers include, but are not limited to, molecules andother cells. Suitable molecules include, but are not limited to, drugs,cytokines, small molecules, hormones, combinations of interleukins,lectins and other stimulating agents, e.g., PMA, LPS, bispecificantibodies and other agents that modify cellular functions or proteinexpression.

[0116] Suitable cells include, but are not limited to, direct cell tocell interactions such as between a tumor and T cell and indirect cellto cell interactions such as those induced by the proximity of othercells which secrete a biological modifier. Suitable cells include, butare not limited to, blood cells, peripheral bone marrow cells andvarious cell lines.

[0117] The incubation conditions are also such that product isessentially not captured or is captured to a much lesser extent byanother cell, so as to distinguish non-producing cells from productproducing cells, or high producers from low producers. Generally theincubation time is between five minutes and ten hours, and is moreusually between one and five hours. The incubation medium can optionallyinclude a substance that slows diffusion of the product from theproducer cell. Substances which inhibit product diffusion in liquidmedia and that are non-toxic to cells are known in the art and include avariety of substances that partially or completely gel, including, forexample, alginate, low melting agarose and gelatin. By varying theviscosity or permeability of the medium, the local capture by aproducing cell of differently sized products can be modulated. Themolecular weight size exclusion of the medium can be adjusted tooptimize the reaction. The optimal composition of the medium can beempirically determined and is influenced by the cell concentration, thelevel of secretion and molecular weight of the product and the affinityof the capture moieties for the product. Such determinations are withinthe skill of one in the art.

[0118] Preferably, the gels are solubilized after the incubation toallow the isolation of the cells or groups of cells from the media bycell sorting techniques. Thus, for example, the gels can be linked bydisulfide bonds that can be dissociated by sulfhydryl reducing agentssuch as β-mercaptoethanol or dithiothreitol, or the gels can contain ioncross-linkings, including for example, calcium ions, that aresolubilized by the addition of a chelating agent such as EDTA.

[0119] At the end of the secretion phase the cells are usually chilledto prevent further secretion, and the gel matrix (if any) issolubilized. This order can, of course, be reversed. As capping can takeplace after the capture moiety is added due to cross linking, anincubation step to decrease capping can be added at this point. Thecells can be incubated for instance in cytochalasin A or B or any othersuitable substance that prevents capping. The cells containing thetrapped product are then labeled with a label moiety. Labeling can beaccomplished by any method known to those of skill in the art. Forexample, anti-product antibodies can be used to directly or indirectlylabel the cells containing the product. The labels used are those whichare suitable for use in systems in which cells are to be analyzed orsorted based upon the attachment of the label moiety to the product.

[0120] In other embodiments, capture moieties that do not containcaptured product can be detected. This allows, for example, theisolation of cells that secrete high amounts by employing a negativeseparation method, i.e., detection of cells not highly saturated withproduct. The cells can be labeled with other labeling substancesrecognizing, e.g., cell surface markers, cell type, cellular parameterssuch as DNA content, cell status, or number of capture moieties.

[0121] The enumeration of actual capture moieties can be important tocompensate for varying amounts of these molecules due to, for example,different conjugation potentials of the cells. It can be especiallyimportant for the isolation of rare cells to exclude cells withdecreased or increased capability for binding the product capturesystem, including the anchor and capture moieties. Alternatively, thereactions can proceed simultaneously in a “one-step reaction.”

[0122] Cell Analysis and Cell Sorting

[0123] Analysis of the cell population and cell sorting based upon thepresence of the label can be accomplished by a number of techniquesknown in the art. Cells can be analyzed or sorted by, for example, flowcytometry or FACS. These techniques allow the analysis and sortingaccording to one or more parameters of the cells. Usually one ormultiple secretion parameters can be analyzed simultaneously incombination with other measurable parameters of the cell, including, butnot limited to, cell type, cell surface markers, DNA content, etc. Thedata can be analyzed and cells sorted using any formula or combinationof the measured parameters. Cell sorting and cell analysis methods areknown in the art and are described in, for example, The Handbook ofExperimental Immunology, Volumes 1 to 4, (D. N. Weir, editor); FlowCytometry Cell Sorting (A. Radbruch, editor, Springer Verlag, 1992); andCell Separation Methods and Applications (D. Recktenwald and A.Radbruch, eds., 1997) Marcel Dekker, Inc. N.Y. Cells can also beanalyzed using microscopy techniques including, for example, laserscanning microscopy, fluorescence microscopy; techniques such as thesecan also be used in combination with image analysis systems. Othermethods for cell sorting include, for example, panning and separationusing affinity techniques, including those techniques using solidsupports such as plates, beads and columns.

[0124] Some methods for cell sorting utilize magnetic separations, andsome of these methods utilize magnetic beads. Different magnetic beadsare available from a number of sources, including for example, Dynal(Norway), Advanced Magnetics (Cambridge, Mass., U.S.A.), Immuncon(Philadelphia, U.S.A.), Immunotec (Marseilles, France), and MiltenyiBiotec GmbH (Germany).

[0125] Preferred magnetic labeling methods include colloidalsuperparamagnetic particles in a size range of 5 to 200 nm, preferablyin a size of 10 to 100 nm. These magnetic particles allow a quantitativemagnetic labeling of cells, thus the amount of coupled magnetic label isproportional to the amount of bound product, and the magnetic separationmethods are sensitive to different amounts of product secretion.Colloidal particles with various specificities are known in the art, andare available, for example, through Miltenyi Biotec GmbH. The use ofimmunospecific fluorescent or magnetic liposomes can also be used forquantitative labeling of captured product. In these cases, the liposomescontain magnetic material and/or fluorescent dyes conjugated withantibody on their surfaces, and magnetic separation is used to allowoptimal separation between nonproducing, low producing, and highproducing cells.

[0126] The magnetic separation can be accomplished with high efficiencyby combining a second force to the attractive magnetic force, causing aseparation based upon the different strengths of the two opposed forces.Typical opposed forces are, for example, forces induced by magneticfluids mixed in the separation medium in the magnetic separationchamber, gravity, and viscous forces induced by flow speed of mediumrelative to the cell. Any magnetic separation method, preferablymagnetic separation methods allowing quantitative separation will beused. It is also contemplated that different separation methods can becombined, for example, magnetic cell sorting can be combined with FACS,to increase the separation quality or to allow sorting by multipleparameters.

[0127] Preferred techniques include high gradient magnetic separation(HGMS), a procedure for selectively retaining magnetic materials in achamber or column disposed in a magnetic field. In one application ofthis technique the product is labeled by attaching it to a magneticparticle. The attachment is generally through association of the productwith a label moiety which is conjugated to a coating on the magneticparticle which provides a functional group for the conjugation. Thecaptured product thus coupled to a magnetic “label”, is suspended in afluid which is then applied to the chamber. In the presence of amagnetic gradient supplied across the chamber, the magnetically labeledtarget cell is retained in the chamber; if the chamber contains amatrix, it becomes associated with the matrix. Cells which do not haveor have only a low amount of magnetic labels pass through the chamber.

[0128] The retained cells can then be eluted by changing the strengthof, or by eliminating, the magnetic field or by introducing a magneticfluid. The selectivity for a captured product is supplied by the labelmoiety conjugated either directly or indirectly to the magnetic particleor by using a primary antibody and a magnetic particle recognizing theprimary antibody. The chamber across which the magnetic field is appliedis often provided with a matrix of a material of suitable magneticsusceptibility to induce a high magnetic field gradient locally in thecamber in volumes close to the surface of the matrix. This permits theretention of fairly weakly magnetized particles. Publications describinga variety of HGMS systems are known in the art, and include, forexample, U.S. Pat. No. 4,452,773, U.S. Pat. No. 4,230,685, PCTapplication WO85/04330, U.S. Pat. No. 4,770,183, and PCT/EP89/01602;systems are also described in U.S. Pat. Nos. 5,411,863; 5,543,289;5,385,707; and 5,693,539, which are commonly owned and herebyincorporated herein by reference.

[0129] In addition, in other embodiments the processes include labelingthe cells that contain the product captured by the capture moiety, ifany. Other embodiments can also include analyzing the cell population todetect labeled cells, if any, and if desired, sorting the labeled cells,if any.

[0130] Diagnostic Methods for Detecting Antigen-Specific T Cells

[0131] The present invention further provides diagnostic methods fordetecting antigen-specific T cells. These include methods for analyzinga population of cells enriched for T cells to identify or enumerateantigen-specific T cells, as well as methods of determining adistribution of antigen-specific T cells that secrete a product inresponse to antigen stimulation.

[0132] Methods for analyzing a population of cells enriched in T cellsto identify or enumerate antigen-specific T cells that secrete andrelease an amount of product relative to other cells in the population,wherein the product is secreted and released in response to antigenstimulation, comprise the steps of labeling the cells by the methods ofthe present invention; labeling the cells with at least one additionallabel that does not label the captured product; and detecting the amountof product label relative to the additional label. Such methods areuseful, for example, in determining the proportion of a cell populationthat is specific for a given antigen. The method can be used to provideinformation regarding the immune status of an individual, includingassessing an immune response to allergens, a tumor or virus, orevaluating the proportion of cells in an individual that are selfreactive so as to detect or monitor autoimmune diseases.

[0133] Method of Treatment Using Enriched Antigen-Specific T Cells

[0134] The present invention provides methods of treatment of a diseaseor condition related to a population of antigen-specific T cells, usingthe enriched T cells of the invention.

[0135] Treatment methods include those in which an antigen-specific Tcell population is identified, enriched, and introduced into anindividual; those in which a population of antigen-specific T cells isidentified, enriched and expanded in vitro before introduction into anindividual; those in which a population of antigen-specific T cells isidentified and eliminated from a population of cells to be introducedinto an individual; ex vivo genetic modification prior toadministration; and selection of antigen-specific T cells selectedaccording to cytokine expression. Examples of antigen-specific T cellsselected according to cytokine expression include, but are not limitedto, IFN-γ or TNF-α secreting CD8⁺ T cells (cytotoxic) for treatment ofcancer, viral (e.g. CMV, EBV) and bacterial (e.g. listeria,mycobacteria) infections; IFN-γ secreting CD4⁺ T cells for the sameindications and also for suppression and/or counter-regulation ofallergy or vaccination against allergy, suppression of TH2-associatedautoimmune diseases or vaccination against these autoimmune diseases;IL-10 or TGF-beta secreting CD4⁺ T cells, for suppression TH1, but alsoTH2-associated autoimmune diseases or vaccination against theseautoimmune diseases (tolerance induction); IL-4 secreting CD4⁺ T cellsfor suppression of TH1-associated autoimmune diseases or vaccinationagainst these autoimmune diseases; and IL-4 or IL-5 secreting CD4⁺ Tcells for treatment of helminth infections.

[0136] T cell populations enriched according to the methods of thepresent invention can be used to treat a variety of disorders. Includedamong these are cancer. T cells specific for a tumor antigen can beobtained using the methods of the present invention. Tumor cells can beobtained from an individual, and these can be co-cultured in vitro withT cells obtained from the same individual. After co-culturing the cellsfor a suitable time, tumor-specific T cells can be enriched accordingthe methods of the present invention. This enriched population can thenbe re-introduced into the patient. Methods for anti-tumor immunotherapyusing autologous T cells are known in the art. See, for example, WO97/05239.

[0137] Alternatively, cells used in anti-tumor immunotherapy treatmentscan be allogeneic. Various modes of treatment of cancer with allogeneicT cells have been described in the art and can be used in the methods ofthe present invention. See, for example, PCT Publication No. WO96/37208. Optionally, allogeneic T cells can be activated prior tointroduction into an individual. Activation can be effected throughcontact with a biological modifier, an antibody directed to a cellsurface marker, or a ligand or analog thereof for a cell surfacereceptor.

[0138] Another use of enriched T cell populations of the presentinvention is in immunomodulation, for example, in the treatment ofautoimmune disorders, inflammatory disorders, allergies andhypersensitivities such as delayed-type hypersensitivity and contacthypersensitivity. T cells which are capable of destroying or suppressingthe activity of autoreactive cells can be enriched in vitro, optionallyexpanded in vitro, then re-introduced into a patient. In the treatmentof allergic responses, the ratio of TH1 to TH2 cells can be altered, or,cells reactive toward allergen-specific cells can be enriched andintroduced into an individual.

[0139] Inducing T cell anergy can also be used to treat, ameliorate orprevent allograft rejection thus improving the results of organtransplantation and increasing the range of histotypes to which apatient can be made histocompatible.

[0140] Compositions comprising enriched T cell populations can furtherbe used as vaccines, to prevent or substantially reduce the probabilityof the occurrence of a disease state such as a viral infection,autoimmune disorder, allergic response, cancer, or other disorder, orwill reduce the severity or duration of the disease if subsequentlyinfected or afflicted with the disease.

[0141] The compositions of cells can be administered by any known route,including, but not limited to, intravenously, parenterally, or locally.In the treatment methods of the present invention, enriched T cells areadministered to an individual. The total number of cells, the number ofdoses, and the number of cells per dose will depend upon the conditionbeing treated. Generally, about 10⁶ to 10¹¹ cells are administered in avolume ranging from about 5 ml to 1 liter. The cells can be administeredin a single dose or in several doses over selected time intervals. Ofthe cells being administered, preferably at least about 10%, morepreferably at least about 20%, more preferably at least about 50%, areantigen-specific T cells which secrete a product.

[0142] Kits

[0143] It is contemplated that the reagents used in the detection ofsecretor cells of desired products can be packaged in the form of kitsfor convenience. The kits would contain, for example, optionally one ormore materials for use in preparing gelatinous cell culture medium, themedium to be used for cell incubation for the production of the desiredsecreted product; a product capture system comprised of anchor andcapture moieties; a label moiety; and instructions for use of thereagents. All the reagents would be packaged in appropriate containers.

[0144] The kit can also be formulated to include the following. In thiscase all the reagents are preferably placed in a single vial to whichthe cells are added. At least one antibody which is bispecific for aparticular cell surface structure or anchor moiety and the product. Atleast one label moiety and, optionally, biological modifiers.

[0145] Optionally, the kit can include physiologically acceptablebuffer. Such buffers are known in the art and include, but are notlimited to, PBS with and without BSA, isotonic saline, cell culturemedia and any special medium required by the particular cell type.Buffers can be used that reduce cross-labeling and increase the localproduct concentration around the cells. Buffers can include agents forincreasing viscosity or decreasing permeability. Suitable agents aredescribed herein. The viscosity of the medium can be reduced beforeanalysis by any method known in the art including, but not limited to,dissolution in a physiologically acceptable buffer, dissolving heat,EDTA, and enzymes. In the absence of added medium, cells alreadysuspended in a medium can be directly added to the vial. Suitable cellsuspensions include but are not limited to cell lines and biologicalsamples. Biological samples include, but are not limited to, blood,urine and plasma.

[0146] Additional structures can be added for catching unbound productto reduce cell cross-contamination thereby reducing the diffusion ofproducts away from the producing cells. These include, but are notlimited to, anti-product antibody immobilized to gel elements, beads,magnetic beads, and polymers.

[0147] Biological modifiers can also be added to the buffer or medium toinduce specific secretion.

[0148] Additional label moieties such as antibodies (magnetically orfluorescently labeled) can also be present, including, but not limitedto anti-cell surface marker antibodies to identify cell types, propidiumiodide to label dead cells, and magnetic beads to label certain celltypes.

[0149] In this embodiment, all materials can be placed in a singlecontainer such as a vial and the cell sample added. The contents areincubated to allow secretion of a product and subsequent capture of theproduct and binding of the label moiety to the product. The cells whichhave secreted and bound product can then be separated and/or analyzedbased on the presence, absence or amount of the captured product.Separation can be done by any of the methods known in the art,including, but not limited to, simple dilution, erythrocyte lysis,centrifugation-washing step, magnetic separation, FACS and Ficollseparation. The analysis of the cells can be performed by a variety ofmethods, including, but not limited to, FACS, image analysis,cytological labeling, and immunoassay.

[0150] The following examples are provided solely for the purposes ofillustration and not to limit the scope of the invention. In light ofthe present disclosure, numerous embodiments within the scope of theclaims will be apparent to those of ordinary skill in the art.

EXAMPLE 1

[0151] Peripheral blood mononuclear cells (PBMC) were cultured incomplete RPMI 1640 (Gibco BRL, Grand Island, N.Y.) containing 100 U/mlpenicillin, 0.1 mg/ml streptomycin, 0.3 mg/ml glutamine, 10 mM2-mercaptoethanol and 10% human serum type AB (Sigma, St. Louis, Mo.) ata cell concentration of 2×10⁶ cells/ml. Peptide M1 58-66 from Influenzavirus matrix protein (GILGFVFTL; Neosystem, Strasbourg, France) wasadded to a final concentration of 1 μM. Control cells were culturedwithout peptide.

[0152] Cells were incubated at 37° C. in an atmosphere containing 7.5%CO₂. After 5 hours and 30 minutes, cells were harvested bycentrifugation. Cells were incubated at a cell concentration of 5×10⁷cells/ml in complete RPMI 1640 with anti human interferon gamma (IFN-γ)monoclonal antibody (mAb) 4SB3 conjugated to anti-human CD45 mAb 5B1 (30μg/ml) at 8° C. for 7 min. The cells were then diluted to 2×10⁶ cells/mlwith complete RPMI 1640 containing 10% FCS and incubated for 45 minutesat 37° C. Then cells were pelleted and incubated with phycoerythrin(PE)-conjugated anti human interferon gamma (IFN-γ) mAb NIB42 (4 μg/ml)and FITC-labeled anti-CD8 mAb in PBS/BSA/EDTA solution 0.05% BSA and 2mM EDTA, for 10 minutes at 4° C. Cells were then washed in PBS/BSA/EDTAand labeled with mouse anti-PE mAb 80-5 conjugated to MicroBeads(Miltenyi Biotec) in PBS/BSA/EDTA for 15 minutes at 8° C. Cells werewashed and resuspended in 500 μl PBS/BSA/EDTA.

[0153] IFN-γ-secreting cells were enriched with the magnetic cellseparation system MACS. Magnetically labeled cell suspension waspipetted onto a MiniMACS separation column in a MiniMACS separationunit, the cell suspension was allowed to pass through and the column waswashed with 3×500 μl buffer. The effluent was collected as negativefraction (N1). The column was removed from the separator, and placed ona suitable tube. 1 ml buffer was pipetted on top of column andmagnetically labeled cells were flushed out using a plunger and appliedto a second round of MiniMACS separation.

[0154] The original cells (i.e., before MACS separation), negative cellfractions (of first as well as second MACS separation, designated N1 andN2, respectively) and positive cell fraction (P2) of second MACSseparation were analyzed by flow cytometry. FACScan and CELLQuestresearch software (Becton Dickinson, Mountain View, Calif.) were usedfor flow cytometric analysis. Dead cells and cell debris were excludedaccording to scatter properties and staining with propidium iodide (PI;0.3 μg/ml).

[0155] The results are shown in FIGS. 1A-P. While dot plots A-H showanalysis of control cells cultured without peptide, plots I-P showanalysis of peptide stimulated cells. Dot plots show-the scatterproperties of the starting cell population (A and I) and the enrichedcell populations (C and K); and PI versus PE fluorescence of thestarting cell population (B and J) and enriched cell population (D andL).

[0156] Dot plots E-H and M-P show anti-CD8-FITC versus anti-IFN-γ-PEstaining of gated cells in original (E and M), first negative (F and N),second negative (G and O) and in the final positive cell fraction (H andP).

[0157] The control cell population, CD8⁺ IFN-γ⁺ cells were enriched upto 11% among live cells (FIG. 1H), in the peptide stimulated cellpopulation, CD8⁺ IFN-γ⁺ cells were enriched up to 40% (FIG. 1P). From astarting population of 3.5×10⁷ control cells, about 600 CD8⁺ IFN-γ⁺cells were isolated, compared to 4100 CD8+IFN-γ⁺ cells isolated from astarting population of 3.5×10⁷ peptide-stimulated cells.

[0158] CD8-cells brightly stained with PE-labeled anti-IFN-γ were CD19⁺B cells, most likely B cells specific for a sorting reagent, probablyPE. These cells were enriched to the same extent from control cellscompared to peptide stimulated cells.

[0159] Also the CD8⁻ cells dimly stained with PE-labeled anti-IFN-γ(like the CD8⁺ IFN-γ⁺ cells) were enriched to the same extent fromcontrol cells compared to peptide stimulated cells. Such cells partiallystain for CD4 and CD56, and therefore are most likely T helper cells orNK cells secreting IFN-γ.

[0160] Thus there is a basal level of IFN-γ secretion by (CD4+) T helpercells, (CD8⁺) cytotoxic T cells and (CD56⁺) NK cells without intentionalantigen-specific stimulation in vitro, which reflects most likely theIFN-γ secretion induced already in vivo in ongoing immune responses atthe time of blood sampling.

[0161] However, IFN-γ⁺-secreting CD8⁺ cells induced by stimulation withthe HLA class I-restricted influenza peptide M1 58-66 were significantlyenriched above this background level; therefore, most of the CD8+IFN-γ⁺cells enriched from peptide stimulated cells are peptide-specific Tcells. Specificity of enriched cells was further confirmed by stainingfor the presence of Vβ17 TCR, which is a conserved T cell receptor (TCR)segment in M1 58-66 specific cytotoxic T cells. Lehner et al. (1995) J.Exp. Med. 181:79-91; and Lalvani et al. (1997) J. Exp. Med. 186:859-865.Among IFN-γ⁺ cells isolated from peptide stimulated cells, but not amongIFN-γ⁺ cells isolated from control cells, most express Vβ17⁺ TCRs.

EXAMPLE 2

[0162] Peripheral blood mononuclear cells (PBMC) were cultured incomplete RPMI 1640 (Gibco BRL, Grand Island, N.Y.) containing 100 U/mlpenicillin, 0.1 mg/ml streptomycin, 0.3 mg/ml glutamine, 10 mM 2-ME and10% human serum type AB (Sigma, St. Louis, Mo.) at 2×10⁶ cells/ml.Peptide M1 58-66 from Influenza virus matrix protein (GILGFVFTL;Neosystem, Strasbourg, France) was added to a final concentration of 1μM. Control cells were cultured without peptide.

[0163] After 5 hours and 30 minutes cells were harvested bycentrifugation. Cells were incubated at 5×10⁷ cells/ml in complete RPMI1640 with anti-human IFN-γ mAb 4SB3 conjugated to anti-human CD45 mAb5B1 (30 μg/ml) at 8° C. for 7 minutes. The cells were then diluted to2×10⁶ cells/ml with complete RPMI 1640 containing 10% FCS and incubatedfor 45 minutes at 37° C. Then cells were spun down and incubated withphycoerythrin (PE)-conjugated anti-human-IFN-γ mAb NIB42 (4 μg/ml) andFITC-labeled anti-CD8 in PBS/BSA/EDTA, for 10 minutes at 4° C. Cellswere then washed in PBS/BSA/EDTA and labeled with mouse anti-PE mAb 80-5conjugated MicroBeads (Miltenyi Biotec) in PBS/BSA/EDTA for 15 minutesat 8° C. Cells were washed and resuspended in 500 μl PBS/BSA/EDTA.

[0164] IFN-γ-secreting cells were enriched with the magnetic cellseparation system MACS. Magnetically labeled cell suspension waspipetted on top of a MiniMACS separation column in a MiniMACS separationunit, cell suspension was allowed to pass through and column was washedwith 3×500 μl buffer. Effluent was collected as negative fraction. Thecolumn was removed from separator, and placed on a suitable tube. 1 mlbuffer was pipetted on top of column and magnetically labeled cells wereflushed out using a plunger and applied to a second round of MiniMACSseparation.

[0165] Original cells (i.e., before MACS separation), negative cellfractions (of first as well as second MACS separation) and positive cellfraction of second MACS separation were analyzed by flow cytometry.FACScan and CELLQuest research software (Becton Dickinson, MountainView, Calif.) were used for flow cytometric analysis. Dead cells andcell debris were excluded according to scatter properties and stainingwith propidium iodide (PI; 0.3 μg/ml) as shown in Example 1. The resultsare shown in FIG. 2.

[0166] While dot plots 2A-G show analysis of control cells culturedwithout peptide, plots 2J-R show analysis of peptide stimulated cells.

[0167] Dot plots 2A-D and 2J-M show FITC-labeled anti-CD8 versusPE-labeled anti-IFN-γ staining of gated cells in original (A, J), firstnegative (B, K), second negative (C, L) and in the final positive cellfraction (D, M).

[0168] While in the control cells CD8⁺ IFN-γ+ cells were enriched up to8.2% among live cells (2D), out of peptide stimulated cells CD8⁺ IFN-γ⁺cells were enriched up to 41.6% (2M). Out of 6.1×10⁷ control cells,about 1360 CD8⁺ IFN-γ⁺ cells were isolated compared to 11700 CD8⁺ IFN-γ⁺cells out of 6.9×10⁷ peptide stimulated cells.

[0169] IFN-γ⁺ secreting CD8⁺ cells induced by stimulation with the HLAclass I-restricted influenza peptide M1 58-66 were significantlyenriched above background level, i.e., most of the CD8⁺ IFN-γ⁺ cellsenriched from peptide stimulated cells must be peptide-specific T cells.Specificity of enriched cells was further confirmed by staining againstVβ17 TCR, which is a conserved T cell receptor (TCR) segment in M1 58-66specific cytotoxic T cells (Lehner 1995; Lalvani 1997). Only amongIFN-γ⁺ cells isolated from peptide stimulated cells, but not amongIFN-γ⁺ cells isolated from control cells, most express Vβ17⁺ TCRs (2Fversus 2O).

[0170] The following examples show that appropriate antigen-specificstimulation, CD4⁺ and CD8⁺ lymphocytes rapidly express cytokines. Thetechnique is demonstrated here for HLA-A0201-restricted influenza matrixprotein (FLU) peptide 58-66 -specific CD8⁺ cytotoxic T lymphocytes(CTLs), influenza A virus- and recombinant tetanus toxin C(rTT.C)-fragment-specific T helper type 1 (Th1) cells, and tetanustoxoid (TT) specific T helper type 2 (Th2) cells.

EXAMPLE 3 Materials and Methods for Examples 4-8

[0171] Cells and Ex Vivo Stimulation

[0172] Buffy coats were obtained from the Institute for Transfusionsmedicine, Hospital Merheim, Cologne, Germany and, if necessary, selectedon the basis of HLA-type. PBMC were prepared by standard Ficoll-Pacque(Pharmacia, Uppsala, Sweden) density gradient centrifugation, washedtwice in PBS and resuspended at a cell concentration of 2×10⁶ cells perml in cell culture medium consisting of RPMI 1640 (Life Technologies,Paisley, UK) supplemented with 10% (wt/vol) human AB-serum (BoehringerIngelheim, Ingelheim, Germany), 1 mM L-alanyl-glutamine (LifeTechnologies), 100 U/ml penicillin/streptomycin (Life Technologies),0.05 mM 2-mercaptoethanol (Life Technologies) and 1 mM sodium-pyruvate(Life Technologies). 12.5 ml of the cell suspension were place in 100×20mm tissue culture dishes (Sarstedt, Newton, Mass.) and FLU 58-66 peptide(Neosystems, Strasbourg, France) was added to a final concentration of 1μM, purified influenza A virus preparation (Biodesign, Kennebunk, Me.)was added to a final concentration of μg/ml, rTT.C (Boehringer Mannheim,Mannheim, Germany) was added to a final concentration of 7 μg/ml andpurified TT (Statens Serum Institut, Copenhagen, Denmark) was added to afinal concentration of 1 μg/ml. Cells were incubated at 37° C. in ahumidified 7.5% CO₂ atmosphere for 5-10 h.

[0173] Capturing of Secreted Cytokines by Cellular Affinity Matrices

[0174] Ab-Ab conjugates directed against CD45 and either IL-4 orIFN-γwere produced by standard protein coupling techniques. Aslam et al.(1998) Bioconjugation, Macmillan Reference Ltd., London. After the exvivo stimulation, cells were harvested using a disposable cell scraper(Costar, Cambridge, Mass.) and labeled for 7 min at a cell concentrationof 10⁸ cells per ml in ice-cold medium with 50 μg per ml of the Ab-Abconjugates. Then, cells were diluted with medium to a final cellconcentration of 2×10⁶ cells per ml and allowed to secrete for 45 min at37° C. in a humidified 7.5% CO₂ atmosphere.

[0175] Magnetic Enrichment and Detection of Cytokine Secreting Cells

[0176] After the cytokine capturing period, cells were harvested again,resuspended at a cell concentration of 10⁸ cells per ml inphosphate-buffered saline containing 0.5% (w/v) bovine serum albumin and5 mM EDTA (buffer) and stained for 10 min at +4° C. with 5 μg/ml antiIFN-γ-PE or anti IL-4-PE, respectively. Cells were washed with buffer(300×g, 10 min), resuspended in 400 μl buffer and magnetically labeledfor 15 min at +4° C. with 100 μl anti PE Ab-microbeads (Miltenyi Biotec,Bergisch, Gladbach, Germany). After washing, the cells were applied ontoa MS+column and placed in a MiniMACS magnet (Miltenyi Biotech). Thecolumn was rinsed with buffer and the retained cells were eluted fromthe column after removing it from the magnetic field to achieve a higherenrichment rate, the eluted cells from the first column were applied toanother MS+column and the magnetic separation was repeated. Cell sampleswere analyzed on a FACScalibur flow cytometer (Becton Dickinson, SanJose, Calif.) using the CellQuest software package.

[0177] Magnetic Enrichment and Detection of Cytokine Secreting Cells

[0178] For detection, enumeration and phenotyping of cytokine-secretingcells the following reagents were used: anti IFN-γ-CD45 (anti IFN-γ,clone 4SB3; CD45, clone 5B1, W. Knapp, Vienna, Austria), anti IFN-γ-PE(clone 45-15), anti IL-4-CD45 (anti IL-4, clone 1 A6-10; CD45, clone5B1, W. Knapp Vienna, Austria), anti IL-4-PE (clone 7A3-3), CD8-Cy5(clone BM135/80, Behring Diagnostics, Marburg, Germany), CD4-Cy5 (cloneM-T321, Behring), CD4-FITC (clone SK3, Becton Dickinson), CD27-FITC(clone M-T271, Pharmingen, San Diego, Calif.), CD28-FITC (clone CD28.2,Pharmingen) CD57-FITC (clone HNK-1, Becton Dickinson), anti Vβ17.FITC(clone E17.5F3.15.13, Coulter-Immunotech, Marseille, France). Meager etal. (1984) Interferon Res. 4:619-625; Alkan et al. (1994) J. Immunoassay15:217-225; and Bird et al. (1991) Cytokine 3:562-567.

[0179] Cytolytic Activity Assay

[0180] The cytotoxic activity of enriched cytokine-secreting cells wasanalyzed using a flow cytometry-based assay which has been describedpreviously. Mattis et al. (1997) J. Immunol. Met. 204:135-142. Briefly,1×10⁶ HLA-A2.1⁺ T2 cells were labeled with 4 μg per ml of the greenfluorescent dye DiO (Molecular Probes, Eugene, Oreg.) inphosphate-buffered saline containing 5 mM EDTA and 3% fetal calf serumfor 45 min at 37° C. Cells were washed three times with buffer,resuspended in cell culture medium and loaded with 1 μM Flu 58-66peptide or Melan A/MART 1 27-35 peptide (Bachem, Heidelberg, Germany)overnight at 37° C. in a humidified 7.5% CO₂ atmosphere. Enrichedcytokine-secreting cells were expanded for 18 d in tissue culture in thepresence of recombinant human IL-2 (Peprotech, London, U.K.). Expandedcytokine-secreting cells and peptide-loaded DiO-labeled HLA-A2.1⁺ T2cells were co-cultivated for 16 h at a ratio of 1:1 at 37° C. in ahumidified 7.5% CO₂ atmosphere. After the culture period, cells wereharvested and analyzed by flow cytometry. In order to permitdiscrimination between live and dead DiO-labeled T2 cells, samples werecounterstained with the red fluorescent exclusion dye propidium iodide.

EXAMPLE 4

[0181] The capability to secrete effector cytokines like IFN-γ followingshort-term antigenic restimulation with synthetic peptide- or nativeantigen-pulsed APCs is a typical feature of memory/effector CD4⁺(Th1-type) and CD8⁺ T cells. Salmon et al. (1989) J. Immunol.143:907-912; and Hamaan et al. (1997) 186:1407-1418. To isolatelow-frequency memory/effector antigen-specific CD4⁺ and CD8⁺ T cellsdirectly from peripheral blood based on antigen-induced secretion ofIFN-γ and cellular affinity matrix technology, peripheral bloodmononuclear cells (PBMC) from HLA-matched adult healthy blood donorswere stimulated for 5-6 h with: (a) the HLA-A0201-restricted FLU peptide58-66, (b) a purified influenza A virus preparation and (c) rTT.C. Afterthe stimulation period, an affinity matrix for IFN-γ was created on thecell surface using antibody (Ab)-Ab conjugates directed against CD45 andIFN-γ, and the cells were allowed to secrete IFN-γ in culture for 45min. Then, IFN-γ, relocated to the affinity matrix of the secretingcells, was stained with a phycoerythrin (PE)-conjugated IFN-γ-specificAb, and PE-labeled cells were enriched by MACS using anti PE Abmicrobeads. See, also, Brosterhus et al., 10th Int. Congress inImmunology, New Delhi, India, 1-6 Nov. 1998, pp. 1469-1473.

[0182] Compared with the non-stimulated control samples, a significantlyhigher proportion of IFN-γ-secreting CD8⁺ cells were detectable afterenrichment in the FLU 58-66 peptide-stimulated sample (FIG. 3A: 38.3%vs. 13.7%), and significantly higher proportions of IFN-γ-secreting CD4⁺cells were detectable after enrichment in the samples stimulated withthe influenza A virus preparation (FIG. 3B: 35.5% vs. 1.1%) and rTT.C(FIG. 3C: 6.1% vs. 0.3%), respectively. When looking at the absolutenumbers of enriched IFN-γ-secreting T cells and their frequencies amongtotal PBMC, differences between the stimulated and non-stimulatedsamples are even more remarkable: (a) 12,500 IFN-γ-secreting CD8⁺ Tcells were isolated from 5.3×10⁷ FLU 58-66 peptide-stimulated PBMC(frequency 1 in 4,200) and 1370 IFN-γ-secreting CD8⁺ T cells wereisolated from 5.1×10⁷ non-stimulated PBMC (frequency: 1 in 37,000); (b)351 IFN-γ-secreting CD4⁺ T cells were isolated from 5×10⁶ influenza Avirus-stimulated PBMC (frequency 1 in 14,000) and 4 IFN-γ-secreting CD4⁺T cells were isolated from 5.0×10⁶ non-stimulated PBMC (frequency 1 in1,250,000); and (c) 132 IFN-γ-secreting CD4⁺ T cells were isolated from1.8×10⁷ rTT.C-stimulated PBMC (frequency: 1 in 136,000) and 7IFN-γ-secreting CD4⁺ T cells were isolated from 1.9×10⁷ non-stimulatedPBMC (frequency: ˜1 in 2,710,000). Considering these experimentalresults, it is evident that IFN-γ-secreting T cells present atfrequencies of below 10⁻⁶ can be detected with our technique.

EXAMPLE 5

[0183] Both memory-and effector-type CD8⁺ T cells are capable ofsecreting IFN-γ. Hamann et al. (1997). To determine the phenotype of FLU58-66 peptide-specific CD8⁺ T cells, enriched IFN-γ-secreting CD8⁺ Tcells from the FLU 58-66 peptide-stimulated sample and the controlsample were analyzed by three-color immunofluorescence for theexpression of a panel of leukocyte surface markers that allow todistinguish between memory and effector-type CD8⁺ T cells. Hamann et al.As shown in FIG. 2, most FLU 58-66 peptide-specific CD8⁺ T cells were(1997) CD27⁺, CD28⁺ and CD57⁻, consistent with a memory phenotype,whereas most of the IFN-γ-secreting CD8⁺ T which became isolatedindependent of the FLU 58-66 peptide were CD27⁻, CD28⁻, CD57⁺,consistent with an effector phenotype. The latter could have beeninduced in vivo to secrete IFN-γ and thus might reflect ongoing immuneresponses.

[0184] More than 54.8% of the IFN-γ-secreting CD8⁺ T cells from the FLU58-66 peptide-stimulated sample expressed the Vβ17 TCR chain, comparedwith less than 2.2% of the IFN-γ-secreting CD8⁺ T cells from the controlsample (FIG. 4). This confirms previous reports showing a bias ofHLA-A0201-restricted FLU peptide 58-66-specific CD8⁺ T cells towards theuse of Vβ17 TCR chain, first in cloned CTLs and later, using fluorescenttetramers of FLU 58-66 peptide-loaded HLA-A2.1 molecules, also in PBMC.Lehner et al. (1995) J. Exp. Med. 181:79-91; and Dunbar et al. (1998).

EXAMPLE 6

[0185] To further confirm the specificity of the enrichedIFN-γ-secreting CD8⁺ T cells from the FLU 58-66 peptide-stimulated PBMC,and to study their cytolytic activity, the cells were expanded for 18 din tissue culture in the presence of IL-2, and then assayed for CTLactivity at an effector: target ratio of 1:1. As shown in FIG. 5,significant killing was observed when target cells were loaded with FLU58-66 peptide, but not when target cells were loaded with a controlpeptide (Melan A/MART 1 27-35).

EXAMPLE 7

[0186] PBMC from 49 HLA-A2+ individuals were cultured with or withoutthe FLU 58-66 peptide and subjected to the enrichment procedure forIFN-γ-secreting cells as described in Example 3. In 45 cases, on averageabout 80-fold more IFN-γ-secreting CD8⁺ T cells were isolated from theFLU 58-66 peptide-stimulated sample as compared to the control sample.Only in three cases, no significant difference was detected between bothsamples. The median frequency of FLU 58-66 peptide-specific CD8⁺ T cellsamong PBMC, as determined by subtracting the frequencies of the controlsamples from the frequencies of the FLU 58-66 peptide-stimulatedsamples, was 1 in 30,000 (range between 1 in 600,000 and 1 in 1000).These results are completely consistent with previous reports in whichthe frequencies of FLU 58-66 peptide-specific CD8⁺ T cells weredetermined using enzyme-linked immunospot (ELISPOT) assays for singlecell IFN-γ release or tetramers of FLU 58-56 peptide-loaded HLA-A2.1molecules. Lalvani et al. (1997; and Dunbar et al. (1998).

EXAMPLE 8

[0187] To demonstrate that our approach isolates live antigen-specificTh2-type CD4⁺ T cells, PBMC were stimulated with purified TT andIL-4-secreting CD4⁺ T Cells were isolated using an Ab-Ab conjugatedirected against CD45 and IL-4. After 10 h of TT stimulation, 150IL-4-secreting CD4⁺ T cells could be isolated from 2.2×10⁷ PBMC with apurity of 6,89% (FIG. 6). This corresponds to a frequency of TT-specificTh2 cells among total CD4⁺ T cells of 1 in 94,000. The frequency ofIL-4-secreting CD4⁺ T Cells in the control culture without TT was about10 times lower.

[0188] All references cited herein, both supra and infra are herebyincorporated herein. Although the foregoing invention has been describedin some detail by way of illustration and example for purposes ofclarity and understanding, it will be apparent to those skilled in theart that certain changes and modifications can be practiced. Therefore,the description and examples should not be construed as limiting thescope of the invention, which is delineated by the appended claims.

1. A method for obtaining a cell population enriched in antigen-specificT cells, comprising the steps of: a) obtaining a mixed population ofcells comprising T cells; b) exposing the cell population to at leastone antigen under conditions effective to elicit antigen-specificstimulation of at least one T cell and allowing expression of at leastone product by the stimulated T cell, wherein the product is secreted inresponse to antigen stimulation; c) modifying the surface of the cellsto contain a capture moiety specific for the product such that thecapture moiety is coupled to the cell surface; d) culturing saidpopulation under conditions wherein said product is secreted, releasedand specifically bound to the capture moiety, thereby labeling theproduct-secreting cells; and e) separating the cells according to thedegree to which they are labeled with said product to obtain apopulation of cells substantially enriched in antigen-specific T cells,wherein steps (b) and (c) can be performed in any order.
 2. A methodaccording to claim 1, further comprising the step of labeling theproduct prior to separation.
 3. The method according to claim 2 whereinthe product is labeled with a label moiety.
 4. The method according toclaim 3 wherein the label moiety is an antibody specific for theproduct.
 5. The method according to claim 3 wherein the label moiety isfluorochromated and the separation is conducted by cell sorting.
 6. Themethod according to claim 3 wherein the label moiety is magnetizable andthe separation is conducted in a magnetic field of sufficient strengthto magnetize the label moiety.
 7. The method according to claim 6wherein the label moiety comprises colloidal magnetic particles with atypical diameter of about 5 to 200 nm.
 8. The method according to claim1 wherein the capture moiety is an antibody or an antigen-bindingfragment thereof.
 9. The method according to claim 8 wherein theantibody or antigen binding fragment thereof is bispecific.
 10. Themethod according to claim 1 wherein the coupling is through a lipidanchor attached to the capture moiety optionally through a linkingmoiety.
 11. The method according to claim 1 wherein the coupling isthrough an antibody or an antigen-binding fragment thereof attached tothe capture moiety, optionally through a linker.
 12. The methodaccording to claim 1 wherein the coupling is through direct chemicalcoupling of the capture moiety to components on the cell surface,optionally through a linker.
 13. The method according to claim 9 whereinthe coupling is through specific binding of the antibody to the cell.14. A method to label antigen-specific T cells with a product secretedand released by the cells, wherein the product is secreted in responseto antigen stimulation, which method comprises: exposing the cells to atleast one antigen under conditions effective to elicit antigen-specificstimulation of at least one T cell; and modifying the surface of thecells to contain a capture moiety specific for the product; andculturing the cells under conditions wherein the product is secreted,released and specifically bound to the capture moiety, thereby labelingthe product-secreting cells.
 15. The method according to claim 14wherein the product is labeled with a label moiety.
 16. The methodaccording to claim 15 wherein the label moiety is an antibody.
 17. Themethod according to claim 14 wherein the capture moiety is an antibodyor an antigen-binding fragment thereof.
 18. The method according toclaim 17 wherein the antibody is bispecific.
 19. The method according toclaim 14 wherein the coupling is through a lipid anchor attached to thecapture moiety optionally through a linker moiety.
 20. The methodaccording to claim 14 wherein the coupling is through an antibody or anantigen-binding fragment thereof attached to the capture moietyoptionally through a linker.
 21. The method according to claim 18wherein the coupling is through specific binding of the antibody to thecell.
 22. A composition obtained from the method according to claim 21.23. The composition according to claim 21 wherein the capture moiety isan antibody or an antigen-binding fragment thereof.
 24. The compositionaccording to claim 23 wherein the antibody is bispecific.
 25. Thecomposition according to claim 22 wherein the coupling is through alipid anchor moiety attached to the capture moiety optionally through alinking moiety.
 27. The composition according to claim 22 wherein thecoupling is through an antibody or an antigen-binding fragment thereofattached to the capture moiety, optionally through a linker.
 28. Thecomposition according to claim 25 wherein the coupling is throughspecific binding of the antibody to the cell.
 29. Cells and progenythereof separated according to the method of claim
 1. 30. Cellsseparated according to the method of claim
 1. 31. A method of analyzinga population of cells to identify or enumerate antigen-specific T cellsthat secrete and release an amount of product relative to other cells inthe population, wherein the product is secreted in response to antigenstimulation, the method comprising the steps of: labeling the cells bythe method according to claim 14, labeling the cells with at least oneadditional label that does not label the captured product, and detectingthe amount of product label relative to the additional label.
 32. Amethod of determining a distribution of secretory activity in a cellpopulation enriched in T cells, the method comprising the steps of:labeling cells by the method according to claim 14, and determining theamount of product label per cell, wherein the product is secreted andreleased in response to antigen stimulation.
 33. The method according toclaim 14 further comprising the steps of: determining the amount andtype of product label per cell wherein distribution of secreted producttype and secretory activity for each secreted product type in apopulation of cells is determined.
 34. A method for identifyingantigen-specific T cells secreting and releasing at least one product inresponse to antigen stimulation, comprising the steps of: combining amixed population of cells enriched for T cells with at least one first,bispecific, antibody, each antibody, having combining sites specific fora cell surface molecule and at least one product; exposing the cellpopulation to at least one antigen under conditions effective to elicitantigen-specific stimulation of at least one T cell; incubating thecombination under conditions and for a time sufficient to allow thecells to secrete the at least one product; adding at least one labelmoiety; and detecting the at least one label moiety.
 35. The methodaccording to claim 34 further comprising the step of separating thecells secreting the product from the mixed cell population.
 36. Themethod according to claim 34 wherein the cell surface molecule is anaturally occurring cell surface protein.
 37. The method according toclaim 36 wherein the protein is a cell surface marker.
 38. The methodaccording to claim 37 wherein the cell surface molecule is selected fromthe group consisting of CD2, CD3, CD4, CD5, CD8, CD11b, CD26, CD27,CD28, CD29, CD30, CD31, CD38, CD40L, CD45RO, CD45RA, LAG3, T1/ST2, SLAM,Class I MHC molecules, Class II MHC molecules, T cell antigen receptor,and β₂-microglobulin.
 39. The method according to claim 34 wherein theincubation conditions include a high viscosity or gel forming medium.40. The method according to claim 34 wherein the label moiety is anantibody.
 41. The method according to claim 40 wherein the antibodycomprises a detectable label.
 42. The method according to claim 41wherein the label is selected from the group consisting of fluorophores,radioactive isotopes, chromophores and magnetic particles.
 43. Themethod according to claim 40 wherein the label moiety is detected byfluorescence activated cell sorting.
 44. The method according to claim43 wherein the label moiety is detected by a third antibody.
 45. Themethod according to claim 44 wherein the label moiety is coupled todigoxigenin and the third antibody is specific for digoxigenin.
 46. Themethod according to claim 45 wherein the third antibody comprises adetectable label.
 47. The method according to claim 46 wherein the labelis selected from the group consisting of fluorophores, radioactiveisotopes, chromophores, and magnetic particles.
 48. The method accordingto claim 47 wherein the label moiety is detected by fluorescenceactivated cell sorting.
 49. The method according to claim 34 wherein thelabel moiety comprises a magnetizable moiety.
 50. The method accordingto claim 49 wherein the label moiety is detected by a third antibodycoupled to a magnetizable moiety.
 51. A method of treating a disease orcondition related to a population of antigen-specific T cells comprisingadministering to an individual in need thereof an amount of a cellpopulation enriched in antigen-specific T cells effective to treat thecondition.
 52. The method according to claim 51, wherein the conditionis selected from the group consisting of an autoimmune disorder, graftrejection, and an allergic response.
 53. The method according to claim51, wherein the condition is a result of a lack of adequate control ofthe condition by antigen-specific T cells.
 54. The method according toclaim 53, wherein the condition is cancer.
 55. The method according toclaim 53, wherein the condition is an infection.
 56. A kit for use inthe detection of antigen-specific T cells that secrete a product inresponse to antigen stimulation, the kit comprising: a product capturesystem comprised of at least one anchor moiety and at least one capturemoiety; and at least one label moiety.
 57. The kit according to claim56, wherein the capture moiety comprises at least one bispecificantibody having at least one antigen recognition site for at least onecell type and at least one antigen recognition site specific for theproduct.
 58. The kit according to claim 57 wherein the at least onebispecific antibody and the at least one label moiety are in a singlevial.
 59. The kit according to claim 57 wherein the at least onebispecific antibody binds to the cell through a cell surface molecule.60. The kit according to claim 57 wherein the cell surface molecule is anaturally occurring cell surface protein.
 61. The kit according to claim57 wherein the cell surface molecule is a cell surface marker.
 62. Thekit according to claim 61 wherein the cell surface molecule is selectedfrom the group consisting of CD2, CD3, CD4, CD5, CD8, CD11b, CD26, CD27,CD28, CD29, CD30, CD31, CD38, CD40L, CD45RO, CD45RA, LAG3, T1/ST2, SLAM,Class I MHC molecules, Class II MHC molecules, T cell antigen receptor,and β₂-microglobulin.
 63. The kit according to claim 55 wherein theincubation conditions include a high viscosity or gel forming medium.64. The kit according to claim 63 wherein the medium is selected fromthe group consisting of gelatin, agarose, alginate and combinationthereof,
 65. The kit according to claim 57 wherein the label moiety isan antibody.
 66. The kit according to claim 65 wherein the antibodycomprises a detectable label.
 67. The kit according to claim 66 whereinthe detectable label is selected from the group consisting offluorophores, radioactive isotopes, chromophores, and magneticparticles.
 68. The kit according to claim 67 wherein the label moiety isdetected by fluorescence activated cell sorting.
 69. The kit accordingto claim 65 wherein the label moiety is detected by a third antibody.70. The kit according to claim 69 wherein the label moiety is coupled todigoxigenin and the third antibody is specific for digoxigenin.
 71. Thekit according to claim 69 wherein the third antibody comprises adetectable label.
 72. The kit according to claim 65 further comprising abiological modifier.
 73. The kit according to claim 56 furthercomprising a cell-cell cross-contamination reducing capture system.